The potential for unintended consequences of the spread of foreign genes  and of pleiotropic effects following transgene expression may be enhanced in long-lived forest trees. The COST Action FP0905 will focus on four key aspects related to the biosafety of genetically modified trees (GMTs): (a) analyses of the efficiency of existing gene containment strategies to avoid or if not possible to minimize gene flow; (b) facilitate efforts to develop site-specific integration of transgenes in tree genomes to minimize variability of transgene expression and pleiotropic effects, (c) evaluate possible methods to monitor GMTs in the whole production chain, and (d) conduct socio-economic and cost/benefit analyses in relation to the use of GMTs in plantations.Populus spp., Pinus spp., Eucalyptus spp., Betula spp., Castanea spp., Picea spp., etc.) as well as extensive expertise in correlated topics. The information gained should contribute to strengthen the scientific basis for the execution of the EU policy directives related to transgenic trees intended for cultivation in Europe. The main objective of the COST Action is to evaluate and substantiate the scientific knowledge relevant for GMT biosafety protocols by putting together already existing information generated in various European and Non-EU countries as basis for future EU policy and regulation for the environmental impact assessment and the safe development and practical use of GMTs.This Action combines multidisciplinary knowledge generated with transgenic lines of different forest genera  to implement collaboration of scientists.WG 1 - Biological characterization of GMTs: to characterize the GMTs in respect to their genetic and phenotypic features relevant for gene flow, gene containment and gene targeting.WG 2 - Environmental impact assessment and monitoring of GMTs in the whole production chain from plantation to final products: to study environmental risk assessment strategies and monitoring the GMTs along the whole production chain.WG 3 - Socio-economic implications of and recommendations for the use of GMTs: to make socio-economics analyses of the use of GMTs considering the concerns and acceptance by the public, the economic potential for GMTs and R&D efforts to be invested, as well as cost/benefit analyses, and propose recommendations for the use of GMTs.WG 4 - Management of intranet - internet websites and dissemination: through a website (www.cost-action-fp0905.eu), provide science-based information and increase public awareness in the utilization of GMTs in forest plantation and at the same time safeguarding the environmentThe knowledge gained will be summarised in a book as a final output of this Action which will report the state of art of knowledge and research on GMTs with suggestion on how to effectively implement present EU directives on GMO considering the problematic of forest trees and their environmental impacts.The Action started the 12th of April 2010 and it will end the 11th of April 2014. Actually, 23 COST countries  have signed the Memorandum of Understanding (MoU). Seven NON-COST countries  are participating to the Action.With integration of all the countries listed above, the EU COST Action FP0905 is expected to generate important benefits as it also foresees a strong collaboration among R&D bodies and legislative directives. This kind of collaboration will be fundamental, on the one hand, to address policy-making efforts and, on the other hand, to allow the scientific community to discuss to public concerns in a responsible way, particularly concerning socio-economic implications and biosafety issues of transgenic tree plantations.


GRHL2) gene was identified in a large family with nonsyndromic sensorineural hearing loss, so far presenting the only evidence for GRHL2 being an autosomal-dominant deafness gene (DFNA28). Here, we report on a second large family, in which post-lingual hearing loss with a highly variable age of onset and progression segregated with a heterozygous non-classical splice site mutation in GRHL2. The c.1258-1G>A mutation disrupts the acceptor recognition sequence of intron 9, creating a new AG splice site, which is shifted by only one nucleotide in the 3′ direction. cDNA analysis confirmed a p.Gly420Glufs*111 frameshift mutation in exon 10. © 2013 Wiley Periodicals, Inc.More than 10 years ago, a c.1609_1610insC mutation in the grainyhead-like 2 ( GRHL2 (OMIM: 608576) with the alias TFCP2L3 (transcription factor cellular promoter 2-like 3), which is a widely expressed transcription factor in human epithelial tissues [Werth et al., GRHL2 spans approximately 177 kb on chromosome 8q22.3 (NCBI 37/hg19) and contains 16 exons, which translate into a 625 amino acid protein. It was first associated with the DFNA28 locus through mapping studies involving a five-generation North American family affected with mild to moderate post-lingual progressive bilateral sensorineural hearing loss. In this family, affected members had a heterozygous c.1609_1610insC mutation in exon 13 [Peters et al., GRHL2 have been associated with marginal significance with age-related hearing impairment susceptibility [Van Laer et al., GRHL2 is not a bonafide deafness gene.Autosomal dominant nonsyndromic hearing impairment accounts for approximately 20% of hereditary hearing loss. To date, there are 54 autosomal dominant loci with 27 associated causative genes identified [Van Camp and Smith, GRHL2 have previously been investigated in animal studies. Northern blot and in situ hybridization studies in the mouse demonstrated high Grhl2 expression in the cochlear duct at embryonic day 18.5 and postnatal day 5 [Peters et al., Grhl2−/− knockout mice were embryonic lethal, displaying split face and neural tube defects. Grhl2+/−/Grhl3+/− compound heterozygotes were viable and exhibited neural tube defects of varying severity. Evidently, coordinated expression of GRHL transcription factors in the non-neural ectoderm is important for neural tube closure [Rifat et al., Tol2 transposon-mediated insertional mutagenesis in zebrafish produced offspring with enlarged otocysts, reduced or absent otoliths, malformed semicircular canals, insensitivities to sound stimulation, and abnormal swimming position despite the normal appearance of hair cells in the inner ear. Upon wild type human GRHL2 mRNA injection, the inner ear defects in the zebrafish were rescued, whereas injection with mutant human GRHL2 was unable to rescue otic defects [Han et al., The expression and function of The study was approved by the Ethics Committee of the University of Würzburg.Genomic DNA was extracted from whole blood using a standard salt extraction method, and was submitted to Otogenetics Corporation  for exome capture (targeting 80 known deafness genes) and next generation sequencing (NGS) on a HiSeq2000 . Paired-end reads of 90–100 bp were analyzed for quality, exome coverage, and exome-wide SNP/InDels using the platform provided by DNAnexus , to which we applied our systematic analysis beginning with the removal of calls that did not meet certain quality and confidence thresholds. Intronic variants not predicted to affect splicing or regulation were also removed, since they are not likely to impact protein structure and function. As we expected the causative dominant mutation to be absent in the healthy population, it is unlikely to be reported in variant databases such as dbSNP and SwissVar. We also used SIFT [Ng and Henikoff, GRHL2 c.1258-1G>A mutation was PCR amplified from genomic DNA using standard PCR cycling conditions with forward primer 5′-GGATTTCACTGGTTTAGGG-3′ and reverse primer 5′-AGCGTAGACTTCAAGTGAGC-3′ . PCR products were sequenced with an ABI 3130xl 16-capillary sequencer .To validate the identified mutation, an amplicon containing the GRHL2 region of interest was amplified using standard PCR cycling conditions from the synthesized cDNA with forward primer 5′-GGAAATACTGGCACTCTCG-3′ and reverse primer 5′-ACCTTCTCGTTCATCATCC-3′. A second round nested PCR continued with inner forward primer 5′-CCGTGAATTGCTTGAGCACA-3′ and reverse primer 5′-GGTTTGCAAAGTGAACATCAG-3′ in order to shorten the amplicon length for Sanger sequencing and enhance the product on the agarose gel.RNA samples were isolated from saliva using a standard protocol from the Oragene RNA collection kit . RNA quality and quantity were assessed with a NanoDrop spectrophotometer . cDNA was produced using the SuperScript III First-Strand Synthesis SuperMix RT-PCR kit . The The index patient (IV:4) developed type I diabetes at the age of 10 and bilateral progressive hearing loss at the age of 32. He does not show any syndromic features and maintains an academic position. The family history allowed for the tracing back of hearing status over five generations . Ten famThe hearing loss in the family members with detailed clinical examination is characterized as bilateral and progressive, usually beginning in the fifth decade of life  with the earliest documented age of diagnosis being 32 years of age in the index patient (IV:4) and latest diagnosis at age 65 (III:3). However notably, several more distantly related individuals without detailed clinical records  had post-lingual childhood onset reported. Apart from childhood onset hearing loss, III:6 had a reduced IQ and severe epilepsy, which were thought to represent comorbidities contributing to his early death at approximately 40 years of age. Individuals III:8, III:10, and III:11 presented with varying spectrums of communication disorders in addition to childhood onset hearing loss and died prematurely at approximately 50 years of age. III:8 was described as having profound hearing impairment beginning from childhood and had infantile seborrhoeic dermatitis. III:10 was reported as having profound hearing loss that made it nearly impossible to communicate with him. III:11 was able to communicate orally with others, but overcompensated for his hearing loss by utilizing stilted speech.Although great care was taken to record the hearing statuses of distantly related family members, there were three individuals having ambiguous hearing classifications. I:2 died at approximately 50 years of age at a time when his affection status was not clear. I:5 was reported as being hearing impaired; however, detailed information about onset and severity was unknown. Both these individuals lived in the 19th century, limiting clinical information to what members of the family collected. II:8 had normal hearing early in life but adult onset hearing loss cannot be excluded. III:12 was reported to hear normally, while his daughter IV:8 had mild hearing impairment at approximately 50 years of age. Thus, III:12 may be a non-penetrant mutation carrier. Unfortunately, III:14 and IV:8 were not available for genetic analysis and, thus, we could not test whether the same form of hearing loss segregates in the left and the right side of the pedigree.Excluding individual IV:2, there was a positive correlation of hearing loss progression and advancing age exceeding what can be expected by normal aging . We perfGJB2 (OMIM: 121011) gene. He was included in a microarray screen of 50 GJB2 mutation-negative non-syndromic hearing loss patients, which did not identify any potentially pathogenic copy number variations (data not shown). We then used targeted deafness gene enrichment sequencing (Otogenetics Corporation) to screen for mutations in 80 known deafness genes including 23 DFNA genes, 32 DFNB genes, and 2 DFN genes (with a number of genes being classified as being both dominant and recessive). Syndromic deafness genes were also included. The analysis strategy we employed filtered out apparently non-pathogenic variants, disclosing a single heterozygous c.1258-1G>A substitution in the GRHL2 gene as pathogenic. The average coverage of GRHL2 in the analyzed data set was 195x. It is worth emphasizing that mutations in the gene responsible for Wolfram syndrome (WFS1), which is characterized by juvenile diabetes mellitus, optic atrophy and progressive hearing loss, were excluded, as WFS1 is also covered in the NGS deafness panel. To date, we have analyzed 24 additional GJB2 mutation-negative hearing loss patients and eight normal hearing controls using targeted deafness gene sequencing and did not find any additional mutation in GRHL2 (data not shown).The index patient was negative for mutations in the GRHL2 mutation did not report hearing loss at the age of 44. In the youngest generation, we identified one heterozygous individual (V:1) and two individuals (V:2 and V:3) without the mutation. Hearing was normal in all three of these individuals, which was expected, considering their young age.Sanger sequencing of genomic DNA (accession: NG_011971.1) showed that the mutation was detected in five family members  with middle to late adulthood onset of hearing loss and absent in the normal hearing father (III:1) of the index patient . IndividGRHL2 intron 9 would result in the skipping of exon 10. To test this, we extracted RNA from saliva samples from the index patient and a normal hearing control, synthesized cDNA (accession: NM_024915.3), and amplified a region spanning exons 9 and 11. Comparing the product size of the patient and control through gel electrophoresis, it was demonstrated that the exon 10 was not skipped  splicing mutation, which could have been misinterpreted as a classic (type I) splice defect if cDNA was not analyzed [Eng et al., This newly identified GRHL2 exon 10 MaxENT score of the 3′ site is 10.1 and the 5′ is 7.4. The cryptic 3′ splice site generated by the c.1258-1G>A mutation has a strong MaxENT score of 9.5. A 6% decrease compared to the wild-type is most likely not sufficient to cause exon skipping or other aberrant splicing, especially since MaxENT scores become increasingly negative as splicing becomes less likely to occur. This further substantiates our cDNA sequence analysis.The maximum entropy model called MaxEntScan by Yeo and Burge  providesGRHL2 is predicted to have a high probability of exhibiting haploinsufficiency. It is plausible to assume that the hearing loss in the present family and the previously reported family [Peters et al., GRHL2 haploinsufficiency.GRHL2 participates in the differentiation and maintenance of epithelial cells throughout life [Werth et al., GRHL2 have progressive, bilateral hearing loss with a typical onset in middle to late adulthood. The variability in the onset of hearing loss and audiometric profiles in heterozygotes argue for the interplay of other genetic or environmental factors in determining the events leading to hearing loss. Comorbidities independent of hearing loss such as epilepsy, reduced IQ, and type I diabetes may influence the onset and severity of hearing loss and explain this variation. Alternatively, given the enormous genetic heterogeneity of hearing loss and the high rate of marriage among hearing-impaired individuals, it is possible that the family members with a documented childhood onset in the right branch of the pedigree, who were not available for genetic diagnostics and detailed clinical examination, suffer from a distinct form of dominant deafness.The results show that the heterozygotes for the c.1258-1G>A mutation in


The future rehabilitation of adults with hearing loss is likely to involve online tools used by individuals at home. Online tools could also be useful for people who are not seeking professional help for their hearing problems. Hearing impairment is a disability that increases with age, and increased age is still associated with reduced use of the Internet. Therefore, to continue the research on online audiological rehabilitative tools for people with hearing loss, it is important to determine if and to what extent adults with hearing loss use the Internet.To evaluate the use of the Internet and email in a group of adults with hearing loss and to investigate if their use of Internet and email differed between genders, among different age groups, and how it compared with the general population in Sweden.Questionnaires containing multiple-choice questions about Internet access, email use, and educational level were mailed to individuals with hearing loss, who were registered as patients at a hearing aid clinic. Out of the 269 invited participants, 158 returned a completed questionnaire, which was a response rate of 58.7%.P<.001). More men than women used the Internet . Use of the Internet was higher in the youngest age group (25-64 years) compared to the oldest age group . A higher usage of the Internet was observed in the participants with hearing loss, especially the elderly, when compared with the general population of Sweden .The results showed that 60% (94/158) of the participants with hearing loss used computers and the Internet. The degree of hearing loss in the group of participants did not explain the level of Internet usage, while factors of age, gender, and education did (We conclude that the use of computers and the Internet overall is at least at the same level for people with hearing loss as for the general age-matched population in Sweden, but that this use is even higher in specific age groups. These results are important for the future work in developing and evaluating rehabilitative educational online tools for adults with hearing loss. The use of the Internet and communication via email, chat forums, blogs, and instant messaging have increased dramatically in western society during recent years. For groups that are suffering from different disabilities, the Internet and the use of online communication tools can be helpful because they can reach out and contact other people without being identified as disabled . ResearcOne of the most common disabilities is hearing loss, and objectively measured hearing loss often occurs prior to subjective hearing difficulties . People Higher age is typically associated with reduced use of the Internet . For exaThe rehabilitation of people with hearing loss, audiological rehabilitation, is a complex process and not always successful. Audiological rehabilitation for people suffering from hearing loss consists of many elements, but most people with hearing loss are not offered any additional rehabilitation once their hearing aids have been fitted. In fact, a large number of adults with hearing loss are not seeking professional help to overcome their hearing problems.Currently, approximately 67% of all Internet users use it to search for online health-related information . In a stThe objective of this study was to evaluate the use of the Internet, computers, and email in a group of adults with hearing loss. We also investigated whether the use of the Internet, computers, and email differed between genders and different age groups and to what degree age, gender, education, and hearing loss could explain the amount of Internet usage. The final aim of our study was to investigate whether there was a difference in the use of the Internet, computers, and email between the general population of Sweden  and a grIn this study, we used systematic sampling by inviting a selected group of hearing aid users from the University Hospital in Linköping, Sweden. Every fourth person who had finished hearing-aid rehabilitation at the University Hospital during 2008 and who did not meet our exclusion criteria was asked to participate in the study via invitation letters sent by regular mail. The defined exclusion criteria were if the potential participant was unable to communicate in Swedish  or was under the age of 18 years. During 2009, invitation letters were sent by mail to a total of 269 individuals. The average age of the invited participants was 73.4 years . The invited participants included 154 out of 269 men (57.2%) and 115 out of 269 women (42.8%).P=.39).Among the invited participants, 173 out of 269 individuals returned their questionnaires (response rate 64.3%), and 158 out of 269 (58.7%) returned a completed questionnaire. We did not send out reminders to the invited participants. The age of the participants who returned a complete questionnaire ranged from 31-96 years . Of the included participants, 96 out of 158 were men (60%) and 62 out of 158 were women (40%). The majority, 85 out of 158 participants (54%), had completed 9 years of elementary school. The lowest number of individuals, 31 out of 158 (20%), had finished 12 years in school and 42 out of 158 (27%) had a university-level degree and therefore more than 12 years in school, as shown in r=-0.46, P<.001), indicating that the degree of hearing loss increased with increasing age, as expected.Measurements of the participants’ hearing losses, as measured by the pure-tone air-conducted hearing thresholds (ISO 8253-1 1989) were collected from their latest visit to the hearing clinic. The pure tone average of the better ear at the four frequencies of 500, 1000, 2000, and 4000 Hz was, on average, 40 dB HL  Do you have a computer at home?, (2) Do you have access to a computer outside your home?, (3) Do you have access to the Internet?, (4) Do you search for information via the Internet?, (5) How often do you use the Internet?, (6) Do you have an email address?, (7) Do you use your email address?, (8) How often do you use your email address?, and (9) What kind of education do you have? see .The medical ethical committee in Linköping, Sweden, approved the protocol.2), and multiple regression analysis were performed. For all analyses, an alpha level of <.05 was used as statistical significance. A multiple regression analysis was used to investigate whether age, education, gender, or hearing loss could predict Internet use and to investigate how much of the variance in Internet use could be explained by age, education, gender, and hearing loss, as shown. The statistical software package Statistica 10, StatSoft, was used.To statistically test the aims of the study, descriptive analysis, Pearson’s chi-square test , had access to the Internet  and used email (P<.001), as shown in P<.001).The second aim was to investigate whether Internet, computer, and email use differed between genders. The results showed that significantly more men than women had a computer (P<.001), as shown in P<.001).The third aim of the study was to analyze the data with respect to age groups to investigate if Internet use and email use were significantly lower in the elderly compared with the younger participants. We divided the participants into three age groups, as shown in Multiple logistic regression analysis was done to investigate whether age, education, gender, or hearing loss could predict Internet use. Multiple logistic regressions were also used to investigate how much of the variance in Internet use could be explained by age, education, gender, and hearing loss, as seen in The final aim was to compare the data generated in this study with the data from a typical Swedish population  in orderP=.06) or computer access at home (P=.10). In age group II (65-74 yrs), there were no significant differences in computer access at home when comparing the two datasets (P=.30), but the participants from the group of adults with hearing loss had significantly more access to Internet than the people in the general population (P=.05). In age group III (75-96 yrs), we found significant differences between the two datasets in both computer access (P=.004) and Internet use (P=.02), which were more common in the group of participants with hearing loss.Additionally, each age group from the two datasets was compared, as shown in The aim of this study was to evaluate the use of the Internet, computers, and email in a group of adults with hearing loss. Furthermore, we investigated if the use of Internet, computers, and email differed between genders and different age groups. The results relating to our first objective indicate that the use of Internet and email is high in the total group of adults with hearing loss, even though it is significantly more common in the younger age groups. We interpret our results of computer and Internet use as valid since the participants reported use of email was nearly the same as computer and Internet use, and we therefore expect that the participants in our sample are using both their computers and their Internet connections. These findings were expected based on the results from earlier studies showing that Internet use in the elderly population is low ,12. HoweIn this study, we also identified a difference due to gender. Our results showed that it was more common for men than women to use computers, the Internet, and email. These findings were expected in this group of participants due to prior research evaluating how gender differences correlate with Internet usage . Our resBecause hearing loss increases with age, the final objective in our study was to evaluate the pattern of Internet use among the elderly and compare with data from the general population of Sweden. More specifically, our aim was to evaluate whether we could detect a higher rate of Internet use among adults with hearing loss. In summary, the results from the multiple regression analysis indicate that Internet use in a group of adults with hearing loss can be explained by age, education, and gender. More specifically, the highest usage of the Internet is seen among the youngest age group and males with higher education levels, and the lowest usage is among the elderly and women with less education. These results are consistent with newly reported findings from Henshaw et al  who inveWhen comparing our data with information technology use in the general Swedish population , we founA reasonable overall interpretation of our findings, together with previous findings, is that people with decreasing hearing sensitivity are using computers and the Internet and also that these media can be useful tools to help the elderly with hearing loss get access to rehabilitation from home ,16,18.In this study, we reported on the use of the Internet, computers, and email in a sample from Sweden, but usage in a similar group in other parts of Europe might be different. The data presented in the study can be seen as a reflection of the present, but the reported use of this type of information technology typically continues to increase over time, especially in elderly people . HoweverThis study was conducted to evaluate whether information technology was used by people with hearing loss in order to explore if developing online rehabilitation tools for them would be fruitful. By using the Internet in the rehabilitation of adults with hearing loss, they could be informed and guided about communication strategies, hearing tactics, ways to handle hearing aids, and other issues in a cost-effective manner. Our results support the idea that, in the near future, the audiological rehabilitation process can be expected to include the Internet and that some elements of rehabilitation and contact with professionals can occur via the Internet using communication tools like email ,18.The conclusion from this study is that, in a systematic, selective sample of hearing aid users, the use of computers, Internet, and email are overall at the same level as the general Swedish population, but this use is even higher in some specific age groups. This information is important for future work in developing and evaluating rehabilitative educational online tools for adults with hearing loss.


Deployment of the air bag in a passenger vehicle accident rarely causes otologic injuries. However, sensorineural hearing loss induced by air bag deployment is extremely rare, with only a few cases reported in the English literature. A 38-year-old man involved in a traffic accident while driving his car at 40 km/hour presented with right sensorineural hearing loss and tinnitus, without associated vertigo. Pure-tone audiometry demonstrated elevated thresholds of 30 dB and 25 dB at 4 kHz and 8 kHz, respectively, on the right side. Air bag deployment in car accidents is associated with the risk of development of sensorineural hearing loss. Passenger-vehicle air bags were invented for protection from various injuries in traffic accidents. Nowadays, vehicles usually come equipped with air bags. These air bags instantly inflate with a large volume of gas in the case of a car collision, creating a brief <100 msec) and intense (150 to 170 dB) pressure wave 00 msec a–5; howevA 38-year-old man was involved in a traffic accident while driving his car at the speed of 40 km per hour. Instantly after the car collision, the air bag deployed in front of him. The patient was found to be conscious and to not have suffered from any damage of the brain. After the accident, the patient began to suffer from fullness and mild tinnitus in the right ear, without associated vertigo. His ear drums were normal, and a tympanogram showed an A-type result. Pure-tone audiometry demonstrated elevated thresholds of 30 dB and 25 dB at 4 kHz and 8 kHz, respectively, on the right side, suggestive of right sensorineural hearing loss. The patient had no history of hearing problems or tinnitus. He was treated by a single intravenous injection of 600 mg of hydrocortisone, followed by oral administration of prednisolone at the starting dose of 40 mg/day, over a 21-day tapering course. The hearing loss recovered partially to 15 dB and 15 dB at 4 kHz and 8 kHz, respectively. The right-sided tinnitus was relieved.Otologic injuries reported to be induced by air bag deployment include tympanic membrane perforation, conductive hearing loss, tinnitus, disequilibrium, and sensorineural hearing loss , 10. SenWe have presented a case of sensorineural hearing loss developing due to air bag deployment in a car accident. Pure-tone audiometry showed elevated thresholds at 4 and 8 kHz. Air bag deployment is associated with the risk of development of sensorineural hearing loss.


Tinnitus is a perception of sound without external source. The exact etiology of tinnitus is not fully understood, although some researchers believe that the condition usually starts in the cochlea. The aim of this study was to determine the potential contribution of outer hair cell dysfunction to chronic tinnitus, by application of Distortion-Product Evoked Otoacoustic Emission (DPOAE) and Transient Evoked Otoacoustic Emission (TEOAE) and also to determine the relationship between tinnitus loudness and the amplitude of these two potentials.This study was conducted on 20 tinnitus patients aged 20–45 years and 20 age- and gender-matched control subjects. DPOAE and TEOAE were performed on each subject. The difference in the amplitudes of TEOAE between the two groups was not significantly different (P=0.08), but the amplitude of DPOAE in patients with tinnitus was significantly lower than the corresponding value in the control subjects (P=0.01). There was no correlation between tinnitus loudness and the amplitudes of neither DPOAE nor TEOAE. Abnormal findings in the DPOAE of tinnitus sufferers suggest some form of cochlear dysfunction in these patients. As there was no correlation between the amplitude of the recorded potentials and tinnitus loudness, factors other than cochlear dysfunction may also influence the loudness of tinnitus.  Tinnitus, from the Latin word tinnire, meaning “to ring,” is a phantom perception of sound when no such sound is present externally . TinnituMany theories have been presented in an attempt to explain the origin of tinnitus in patients with normal hearing. Although tinnitus may be associated with abnormalities at any level of the auditory pathway, it very often starts in the cochlea. Indeed, Jastreboff et al 1993) considers that tinnitus usually starts in the cochlea and generates abnormal activity in the central pathways, which prolongs the systems; the central auditory pathways do not need to be structurally altered . It was 93 considOtoacoustic emissions (OAEs) are a propagation of the audiofrequency signal into the ear canal from the cochlea, transmitted through the ossicular chain and tympanum. They are believed to originate from an active mechanical force generated by the outer hair cells .OAEs are an important tool for the objective evaluation of the inner ear function, especially the outer hair cells of the cochlea. They occur in response to acoustical stimulation, or can even appear spontaneously. Otoacoustic emissions can be evoked by a broadband stimulus (a click) or by brief tones . As otoacoustic emissions are invariably associated with functioning outer hair cells (OHC), their presence is a reliable indicator of cochlear structural integrity, and their absence may indicate a cochlear lesion. Furthermore, by recording otoacoustic emissions, a subclinical cochlear lesion may be detected, as up to 30% of the OHC population may experience damage before any audiometric evidence is apparent in the quarter octaves pure-tone audiometry from 0.125 kHz to 16 kHz. Therefore, we selected this test to evaluate the cochlea in tinnitus patients . The aim of this study was to evaluate cochlear function in tinnitus patients; in other words, we wanted to determine whether or not tinnitus occurs because of a dysfunction at the level of the OHCs. In this study, we used evoked otoacoustic emissions (EOAE) to assess the function of the OHCs in tinnitus patients with normal hearing.Subjects: The patient group consisted of 20 adult males with subjective tinnitus  who had normal hearing thresholds as revealed by pure-tone and speech audiometry measurements who attended an audiology clinic in a university center, from June 2011–July 2012. Normal hearing was defined as a hearing level of 20 dB or less at all audiometric frequencies .  A group of 20 healthy male subjects with normal hearing and no history of tinnitus or auditory or vestibular symptoms participated as a control group. The two populations had similar gender and age distribution. All patients had a definitive diagnosis of subjective tinnitus that lasted more than 1 month, as well as a thorough medical history and clinical examination verified by an otologist. The following exclusion criteria for both the patient and control groups were applied at baseline: history of familial hearing loss, tinnitus perception less than 1 month, use of ototoxic drugs, chronic otitis media, head trauma, and age >45 years.Audiometric Test BatteriesAll subjects underwent the following test battery: pure-tone audiometry, tympanometry  and stapedial muscle reflex, tinnitus pitch-matching procedures, tinnitus loudness-matching procedures, Transient Evoked Otoacoustic Emission (TEOAEs), and Distortion-Product Evoked Otoacoustic Emission (DPOAEs).Subjects were seated in an acoustically test chamber. First, pure-tone audiometry, tympanometry, and stapedial muscle reflex were performed, and then the pitch and loudness of tinnitus was evaluated. In this measurement we asked the patient to compare the pitch produced by a noise or pure tone to the most prominent pitch of tinnitus. They then adjusted the intensity of a noise or tone so that it had the same loudness as their tinnitus. Next we evaluated otoacoustic emission. The first ear examined was the right ear in patients with bilateral tinnitus; while in unilateral patients we first assessed the affected ear. TEOAE testing always was performed first. TEOAEs were performed with a wide band click in a continuous mode and an intensity of 80 dB peak sound pressure level (SPL). For measuring distortion product (DP) grams, the frequency separation of the primaries was f2/f1 = 1.22, with L1 and L2 set to 65 and 55 dB SPL, respectively.th percentile of the normal distribution for the frequencies tested; 3) only the frequencies from 1,000 to 8,000 Hz were considered.The following parameters were considered in the TEOAE tests: 1) signal-to-noise (S/N) ratio_6 dB 2) and reproducibility of the responses of at least 70%. In the DPOAE, the parameters of interest in all tested frequencies were: 1) S/N ratio_6 dB in; 2) amplitude of the signal in the 90Statistical Analysist test, Mann-Whitney U test, and one-way analysis of variance (ANOVA), at the significance level of P<0.05.All data were analyzed using SPSS version 16.0 , including routine statistical procedures such as Leven’s independent Mean (± standard deviation) of age was 29/10 (± 6/43) years for patients and 28/40 (±6/02) years for controls. There was no statistically significant difference between the two groups with respect to gender or age (P>0.05).Pure-Tone AudiometryResults of pure-tone audiometry confirmed normal hearing in all subjects, and there were no significant differences in the mean threshold levels between the groups (P>0.05). The results of audiometric test are shown in .TympanometryTympanometric measures in all subjects were within normal ranges and the middle ear pressure was comparable between the groups.Acoustic ReflexesIn all subjects, acoustic reflexes, ipsilateral and contralateral, were lower than 110 dB across at least three adjacent frequencies.Otoacoustic Emission TestsWe chose TEOAE and DPOAE because they are the most commonly used OAE tests in clinical practice and also they have more standardized methodology. The criteria for normality are stated in the Materials and Methods section. TEOAEsThere was no significant difference between the mean of overall TEOAE amplitude in patients with bilateral tinnitus, unilateral tinnitus, and normal subjects (P>0.05) .DPOAEsThe mean of overall DPOAE amplitudes in patients with tinnitus was significantly lower than the corresponding value in normal subjects (P<0.05); however, there was no difference between patients with unilateral and bilateral tinnitus .Our comparison between the amplitude of DPOAE in tinnitus patients and normal subjects was in the frequency of tinnitus, which we defined through the pitch-matching test for each patient.Correlation Between Tinnitus Loudness and DPOAE Results Pearson’s correlation coefficient was used to evaluate the degree of agreement between tinnitus loudness and DPOAE in the tinnitus patients. There was no correlation between tinnitus loudness and the amplitude of DPOAE in patients with tinnitus (P>0.05).Tinnitus is an otologic symptom and, despite considerable research in this field, the exact pathophysiological processes of tinnitus remain unknown. Involvement of the whole auditory system, either peripheral or central, should be considered in the generation of tinnitus.Some hypothesized pathomechanisms and causes of tinnitus include spontaneous otoacoustic emissions, edge theory, discordant theory, dorsal cochlear nucleus, hyperactivity/plastic readjustment of dorsal cochlear nuclei (DCN), crosstalk theory, pharmacological causes, and neurologic disorders.As the cThe high incidence rate of cochlear damage and studies of cochlear mechanics have led to the suggestion that tinnitus at themed and high frequencies might, in many cases, arise from the cochlea; although the possibility that an acute exacerbation of tinnitus may result from changes in the efferent nervous system has also been suggested . A cochlOur objective was to verify cochlear dysfunction in patients with normal hearing who have tinnitus with the use of the TEOAE and DPOAE tests, and to evaluate the correlation between tinnitus loudness and amplitudes of the recorded potentials. Szutka et al (2010) reported markedly higher DPOAE amplitudes in tinnitus patients without hearing loss and they explained that tinnitus may be caused by increased motility of the OHCs induced by decreasing efferent fiber activity, and not by OHC failure  were more complicated, as they reported that ears in which tinnitus is present exhibited relatively increased amplitudes of DPOAE at high frequencies (4–6.3 kHz) when compared with normal controls, and relatively decreased DPOAE amplitudes at mid frequencies (1650–2400 Hz)  who selected patients from a general tinnitus population and found no difference in TEOAE parameters between patients with tinnitus and normal subjects . However failure . The res2400 Hz) . In addition, Granjeiro et al (2008) reported that patients with tinnitus had a significantly higher prevalence of abnormal TEOAEs and DPOAEs than normal subjects . FernandIn our study, patients with tinnitus showed significantly lower DPOAE amplitudes than subjects who had no auditory complaints, suggesting OHC dysfunction. This observation is in agreement with studies by Ami et al 2008) and Hesse et al (2005), of DPOAE in tinnitus sufferers which showed that reduced OHC activity, as detected by reduced DPOAE levels, may manifest as tinnitus even before there is a shift in hearing threshold ,20. 08 and HeWe assumed that the difference between the results of TEOAE and DPOAE is because the DPOAEs are considered to have a higher sensitivity. Therefore, we recorded DPOAE with a lower amplitude in comparison with TEOAE. We found no relationship between tinnitus loudness and the magnitude of OHC damage, meaning that factors other than cochlear dysfunction may affect tinnitus loudness.Patients with normal hearing who have tinnitus may have cochlear damage. Although referring to a limited population, this observation might be useful in the attempt to elucidate the mechanisms of tinnitus generation and perception. We concluded that DPOAE is a more useful tool in an estimating cochlear damage in tinnitus patients with normal hearing. Further studies with a larger sample size and using other audiometric tests are needed for further evaluation of the mechanism of tinnitus generation.


Emblemasoma auditrix is to locate the host, males of the cicada species Okanagana rimosa, by their calling song. This task is performed by female flies, which deposit larvae into the host. We show that male E. auditrix possess a hearing sense as well. The morphology of the tympanal organ of male E. auditrix is rather similar to the female ear, which is 8% broader than the male ear. In both sexes the physiological hearing threshold is tuned to 5 kHz. Behavioural tests show that males are able to orient towards the host calling song, although phonotaxis often is incomplete. However, despite extensive observations in the field and substantial knowledge of the biology of E. auditrix, no potentially adaptive function of the male auditory sense has been identified. This unique hearing system might represent an intralocus sexual conflict, as the complex sense organ and the behavioural relevant neuronal network is adaptive for only one sex. The correlated evolution of the sense organ in both sexes might impose substantial constraints on the sensory properties of the ear. Similar constraints, although hidden, might also apply to other sensory systems in which behavioural functions differ between sexes.Sensory modalities typically are important for both sexes, although sex-specific functional adaptations may occur frequently. This is true for hearing as well. Consequently, distinct behavioural functions were identified for the different insect hearing systems. Here we describe a first case, where a trait of an evolutionary novelty and a highly specialized hearing organ is adaptive in only one sex. The main function of hearing of the parasitoid fly  Adaptive phenotypes are a result of evolutionary processes underlying natural selection, genetic drift and sexual selection. In sexual reproducing species, such phenotypes have also to be balanced between the sexes In insects hearing evolved several times independently Emblemasoma auditrix parasitizes sound producing males of the cicada Okanagana rimosaO. rimosa for larva deposition into the host. Here, we investigate whether males have an auditory sense and whether the sense organ is functional physiologically and behaviourally. The findings are discussed in respect to the character evolution in both sexes.In addition to the Tachinidae, some species of Sacrophagidae convergently evolved auditory host finding Okanagana rimosa). The pupae were transferred to University of Göttingen, Germany and kept for up to six months at 4°C. Adult flies of both sexes emerged 14 to 20 days after transferring them into room temperature. Adult flies were kept with water and sugar ad libidum and were investigated 3–5 days after emergence.Animals were observed and collected in the field near Grayling or Pellston, Michigan, USA. For electrophysiology pupae were collected from infected host cicadas , Pellston for behavioural tests. Animals had their wings clipped off to prevent escape and were kept in small cages, with sugar and water ad libidum. Flies were tested up to seven days after their capture. Altogether 17 flies were tested, up to three times in an experiment.In the field, male O. rimosa was digitised  and stored on a compact disc . The calling song consisted of chirps with 8–10 kHz peak frequency, 6 ms duration and a repetition rate of 83 chirps per second (cps); cicadas emit the calling song with 90 dB SPL at 10 cm distance The arena for behavioural tests (50 cm x 70 cm) was weakly illuminated from above (300–430 Lux). The arena was covered with cloth to minimise optical cues. One piezo loudspeaker  was placed behind the cloth in the centre of one side. Flies were released in 50 cm distance from the front of the loudspeaker. For phonotaxis experiments the calling song of the host In the field the signals were broadcasted with a sound pressure level of 90–100 dB SPL and animals performing phonotaxis were collected For electrophysiological determination of the hearing threshold the animal was fixed dorsal side up and the neck connective was exposed For scanning electron microscopy of the tympanal organ, the anterior thorax was dissected and fixated in 4% paraformaldehyde. After dehydration, preparations were critical point dried  and sputtered with gold . The preparations were viewed with a Leo 438VP scanning electron microscope and pictures were digitised (1024×768 pixel) with a CCD camera. Additional morphometric measurements were performed with a dissecting microscope (Leica MS5) and a calibrated ocular.Emblemasoma auditrix is not protected and experiments were done in accordance with the regulations for invertebrate research in the US and Germany.For the field work, access to the Pellston site was permitted by the Biological Station of the University of Michigan (UMBS). The Grayling site is public land, no permission is required. Emblemasoma auditrix possesses an ear at the prothorax, directly behind the head  male flies were detected in the vicinity of the experimental setup. More regularly, male flies could be caught early in season by sweeping patches with dry vegetation. At these patches matings have been observed; analyses of the status of mating females showed that they had developed ovaries, but no larvae (n = 3). Recorded sounds at these places included bird vocalizations and environmental noise (wind), but no specific fly related sounds.In the field, female Behavioural experiments in the laboratory clearly demonstrated that male flies react to sound signals. A male was released in the arena and the behaviour in response to the calling song of the cicada was quantified. Complete phonotaxis has not been observed in contrast to females E. auditrix possess an ear, are able to hear and may use it for phonotaxis towards cicada songs. Nevertheless, since the known hearing function (host finding for larvae deposition) is exclusively related to the female, the main question arises: why do males hear at all? We propose that for the species E. auditrix hearing has an adaptive value for the female only, while for the male no detectable function exists. This proposed non-function in one sex can only be revealed with negative evidence, which principally is difficult to produce. In the following we firstly carefully check the male hearing sense against the known functions of hearing in insects The results clearly show that males of Emblemasoma and no specialized sound producing structures have been detected on the flies. Flies may produce buzzing noises during flight, but these noises do not seem to play a role for long range intraspecific signalling: no buzz related auditory behaviour has been detected and the rather high hearing threshold makes such behaviour unlikely. For Diptera in general it is known that Near Field sounds produced by the wing movements may be important for sex recognition in short distance and that such sounds contain frequencies below 1 kHz E. auditrix is a sound pressure receiver, reacting to Far Field sounds of higher frequencies than 1 kHz.This function of insect hearing requires sound production of the species. Evolutionarily, in a taxon like Orthoptera sound production is rather old and it is even more widely distributed than hearing In addition to self-generated sounds, also sound from external sources might be used by both sexes for intraspecific interaction, like gathering together at a sound source. However, screening for environmental sounds in the habitat did not reveal any abiotic sound source which might attract both sexes. Furthermore, non-biological noises are irregular and have frequency spectra below 2 kHz E. auditrix is active during (sunny) daytime It is self-evident that it is advantageous to detect all kinds of stimuli, which would indicate appearance of a potential predator. The male ear, therefore, might serve as a predator detector. However, for what predators might the ear be specifically adaptive? E. auditrix hearing tachinid flies are active during night. Therefore, a predator avoidance function has been suggested for male hearing in Ormia ochraceaE. auditrix it never has been observed that males of Ormiini are attracted to songs of hosts  has been detected. For example, wing beat noises have lower frequencies E. auditrix (peak at 5 kHz). Additionally, sympatric blow fly species do not possess an ear. A well-developed visual sense and a vibration sense seem to be more important for predator avoidance. Like many other flies, E. auditrix reacts sensitive to vibratory stimuli Selective pressure for auditory detection of diurnal predators has not been clearly demonstrated, although some butterflies might be able to detect bird sounds E. auditrix does not support such a hypothesis either: Already in early June nearly all female E. auditrix which were attracted to the loudspeaker were carrying fully developed larvae in their uterus This function is exclusively necessary for females of the investigated parasitoid species. A female is perfectly able to infect a cicada E. auditrix follow fast moving objects as do many other Oestroidea flies By contrast to phonotaxis, visually guided male chasing behaviour and mating have been observed in the appropriate microhabitat. For mating male Therobia leonidei  and seems to be adaptive to ensure an overlap in seasonality of parasitoid and host. Taken together, mate finding in E. auditrix does not rely on the cicada song.Furthermore, the reproductive behaviour of this fly species is related to the seasonality of the host. Adults of the host cicada species emerge from the ground by mid-June E. auditrix. Whereas the hearing system is clearly present in males, the positive behavioural reaction to the host signal, however, was detected by a sensitive scoring system. In the following we discuss the physiology in respect to genetic links which might have led to the evolution of the sense organ in both sexes, although it is needed only in one sex.None of the functions suggested so far for insect hearing seems to be relevant for male E. auditrix since a probably functionless trait has evolved in the context of correlated characters. Ears in both sexes are rather similar, e.g. in respect to tuning to 5 kHz instead of matching the peak frequency of the host's calling song (9 kHz). This mismatch has already been puzzling during interpretation of the female hearing Evolution of traits is balanced between sexes E. auditrix might be due to the evolutionary origin from a precursor chordotonal organ the function of which is still represented in the present ear A non-functional organ in one sex might also lead to sexually dimorphic sense organs or to reduction of the organ. Both processes are observed among hearing insects. In insect species with sexually dimorphic hearing organs divergent functions for the sexes have been found or proposed The developmental genetics for formation of a tympanal ear in insects are not known and hopefully future gene expression studies might reveal the mechanisms for development of the different phenotypes. Nevertheless, possible intralocus sexual conflicts would have implications for interpretation of the traits for hearing and evolution of insect ears in general. For intraspecific acoustic communication, the basic scheme is that one sex  signals and the other sex  responds to the sounds Following the arguments above, we have identified a sense organ that is functional in both sexes but is probably not adaptive in males. This process could only be detected in an example, in which despite careful investigations no - not even a speculative - function was detected. It seems plausible that the hearing sense evolved correlated in both sexes, even though it has a function in one sex only. The evolutionary origin of insect hearing organs also speaks for the hypothesis that the initial traits are genetically coupled and that only later the traits came under the influence of sex-specific adaptations.


H-1,2,4-triazol-1-yl)heptan-3-ol). Here, we carried out a structure-activity relationship study of TIS13 to discover more potent and specific SL biosynthesis inhibitor because TIS13 has a severe side effect at high concentrations, including retardation of the growth of rice seedlings. TIS108, a new TIS13 derivative, was found to be a more specific SL biosynthesis inhibitor than TIS13. Treatment of rice seedlings with TIS108 reduced SL levels in both roots and root exudates in a concentration-dependent manner and did not reduce plant height. In addition, root exudates of TIS108-treated rice seedlings stimulated Striga germination less than those of control plants. These results suggest that TIS108 has a potential to be applied in the control of root parasitic weeds germination.We previously discovered a lead compound for strigolactone (SL) biosynthesis inhibitors, TIS13 (2,2-dimethyl-7-phenoxy-4-(1 Orobanche and Strigamore axillary growth (max) mutants of Arabidopsis, ramosus (rms) mutants of pea, dwarf (d) or high tillering dwarf (htd) mutants of rice and decreased apical dominance (dad) mutants of petunia, revealed that the biosynthesis of SLs is mediated by two carotenoid cleavage dioxygenases, CCD7  and CCD8 , one cytochrome P450 monooxygenase (MAX1) and one novel iron-containing protein (D27) Strigolactones (SLs) are a group of terpenoid lactones that have been found in root exudates of various plant species and were first identified as seed germination stimulants of root parasitic weeds such as Striga in Africa with a loss of $US 7 to 10 billion Root parasitic weeds are considered as harmful plants in sub-Saharan Africa, the Middle East and Asia that parasitize the roots of host plants. It was reported that around 300 million people are affected by Biosynthetic inhibitors of biologically active substances can control their endogenous levels in developmental stages and sites of various plants. In addition, even if a mutation or targeted knockout of an individual gene in sets of paralogous genes could affect phenotypes to a small degree, biosynthetic inhibitors can overcome such gene redundancy in many cases. Therefore, the use of specific biosynthesis inhibitors is an alternative and valuable way to determine the physiological functions of endogenous substances. As was seen for brassinosteroid biosynthesis inhibitors H-1,2,4-triazol-1-yl)heptan-3-ol (TIS13) Striga germination-stimulating activity in root exudates. However, TIS13 treatment strongly reduces plant height that is not caused by the inhibition of SL biosynthesis, suggesting the inhibition of the biosynthesis of other phytohormones such as gibberellin and/or brassinosteroid. To develop specific SL biosynthesis inhibitors, it is necessary to weaken this side effect. In this context, we synthesized several TIS13 analogues and evaluated their ability to inhibit SL biosynthesis and their side effects.To discover new SL biosynthesis inhibitors, we screened a chemical library of triazole derivatives, because several triazole-containing chemicals have previously been shown to act as efficient inhibitors of cytochrome P450 monooxygenases and the proposed SL biosynthesis pathway involves at least one cytochrome P450 (CYP711A). We discovered one SL biosynthesis inhibitor, 2,2-dimethyl-7-phenoxy-4-(1a) to give α-bromoketone (b), which is coupled with triazole under basic conditions to give c, which in turn gave d following aprotic and basic conditions. Subsequent reduction with sodium borohydride gave e.To find specific inhibitors for SL biosynthesis, we performed a structure-activity relationship study. epi-5DS, a major endogenous SL in rice seedlings, in root exudates by LC-MS/MS and measured the length of the second leaf sheath in rice (Oryza sativa L. cv. Shiokari). SL analysis was performed according to a previous report epi-5DS levels in root exudates more effectively than did TIS13, but they were as active as TIS13 in causing dwarfism . We found that both enantiomers were equally effective in reducing the level of epi-5DS in root exudates in comparison with racemic TIS108 (data not shown).To determine the ability of these chemicals to inhibit SL biosynthesis and cause dwarfism, we analyzed the level of dwarfism . These ct growth . TIS101,dwarfism . These rd TIS108 . TIS108 an TIS13 . Eventuaepi-5DS in root exudates, we analyzed endogenous epi-5DS levels in rice roots. The data clearly indicates that TIS108 strongly reduced the levels of epi-5DS in both roots and root exudates in a dose-dependent manner at the concentration ranges of 10–100 nM  biosynthesis. Therefore there is the possibility that TIS108 may affect some of the P450s involved in their biosynthesis pathways and reduce plant height. Rice is very sensitive to GA biosynthesis inhibitors such as paclobutrazol or uniconazole-P and reduces plant height when treated with these inhibitors at 500 nM TIS108 reduces the level of SL, but its target site(s) is still unknown. Given the fact that at least one P450 (MAX1) is involved in SL biosynthesis in Arabidopsis and that there are five MAX1 homologues in rice, the target site(s) of TIS108 could be MAX1 homologues. However, several steps in the SL biosynthesis pathway still have to be elucidated In this study, we demonstrated that TIS108 is a potent SL biosynthesis inhibitor. However, as there still remains a lot of room for structural modification of this chemical group, further studies on structure-activity relationships will lead us to find more potent and specific SL biosynthesis inhibitors. In addition, the results of this study will contribute to design new SL inhibitors.R*,8bS*,2′R*)-GR24 with the same relative stereochemistry as (±)-strigol.GR24 was synthesized as previously described TIS13 derivatives were synthesized essentially as reported H-1,2,4-trizol-1-yl) hexan-1-one (9 : TIS108): 1-phenyl-2-ethanone (1.3 g) and sodium hydride (0.4 g) were dissolved in dimethylformamide (4 mL) and stirred for 30 min, and then (4-chlorobutoxy)benzene(1.85 g)was added under nitrogen atmosphere and stirred for 12 h at room temperature. The reaction mixture was quenched with distilled water. The aqueous layer was extracted three times with ethyl acetate. The organic layer was dried with NaSO4 and concentrated under reduced pressure. The resulting oil was purified by column chromatography . The product was isolated as a white solid in 12.7%. 1H NMR  δ1.43–1.61 , 1.75–1.90 , 2.15–2.36 , 3.92 , 6.09 , 6.83 , 6.93 , 7.26 , 7.51 , 7.63 , 7.95 , 7.99, 8.38 ; 13C NMR  δ 194.2, 158.8, 151.4, 143.0, 134.4, 134.3, 129.5, 129.2, 128.8, 120.8, 114.4, 67.1, 63.9, 32.7, 28.6, 22.8; HRMS (ES+) calcd. for C20H23N3O2+ (M+H) 336.1712, found 336.1707; Melting Point 107–108°C.6-phenoxy-1-phenyl-2-  as an internal standard. The organic layer was dried under nitrogen and dissolved in 1 ml ethyl acetate:n-hexane . The solutions were loaded onto Sep-Pak Silica 1 ml cartridge , washed with same solution twice, eluted with ethyl acetate:n-hexane  three times and concentrated in vacuo. The roots were homogenized in acetone containing d1-epi-5DS (200 pg). The filtrates were dried under nitrogen and dissolved in water. The solutions were extracted with ethyl acetate twice, dried and dissolved in 10% acetone. The extracts were loaded onto Oasis HLB 3 ml cartridges (Waters), washed with water twice, eluted with acetone twice and dried under nitrogen. The concentrates were dissolved in 1 ml ethyl acetate:n-hexane  and loaded onto Sep-Pak Silica 1 ml cartridge, washed, eluted and concentrated to dryness. The epi-5DS-containing fractions from culture media and roots were dissolved in 50% acetonitrile and subjected to LC/MS-MS analysis.SL analysis was performed according to the previously described method S. hermonthica was performed as described previously Germination assay using


Lawsonia intracellularis bacteria in faeces of growing pigs with different levels of diarrhoea.The objective of this study was to investigate the association between average daily gain and the number of n = 150 pigs) was performed in a Danish herd from day 29 to 47 post weaning. Every third day all pigs were weighed, subjected to a clinical examination and faecal samples were obtained. Faecal samples were subjected to dry matter determination and absolute quantification by PCR for L. intracellularis and porcine circovirus type 2 (PCV2). Association between average daily gain, faecal dry matter content, numbers of L. intracellularis bacteria and PCV2 genome copies in faeces was investigated in a multilevel mixed-effects linear model.A longitudinal field study (L. intracellularis log10 bacteria/g faeces were significantly associated with decreasing average daily gain (P < 0.001). The association was decreasing with increasing faecal dry matter content (P < 0.01). The number of PCV2 log10 copies/g faeces was not significantly associated with average daily gain of the pigs (P > 0.5).Increasing numbers of L. intracellularis in growing pigs. Faecal dry matter content must be taken into consideration in interpretation of such test results.The results suggest a potential application of a PCR quantifying  Lawsonia intracellularis is a common intestinal infection in pigs and of economic significance in the pig industry worldwide[L. intracellularis without reduction in average daily gain (ADG)[L. intracellularis in faeces for eight to 12 weeks[L. intracellularis in faeces is therefore not necessarily related to a clinical problem of for example uneven growth in a group of pigs.orldwide. Pigs main (ADG). Further12 weeks. DemonstL. intracellularis bacteria in mucosal scrapings and the severity of intestinal lesions has previously been demonstrated[L. intracellularis bacteria in faeces could potentially also be associated to severity of clinical signs, pathology and average daily gain. However, since L. intracellularis is an obligate intracellular bacterium and can only be cultivated and maintained in cell cultures[L. intracellularis in faeces[L. intracellularis in faecal samples[L. intracellularis bacteria in faeces on a routine basis. This may be useful for diagnosis of the disease severity in pigs infected with L. intracellularis.Correlation between the number of nstrated. Therefocultures, little n faeces. Resent  samples-10 has nL. intracellularis bacteria in faeces of growing pigs with different levels of diarrhoea under field conditions.The objective of the study was to investigate the association between ADG and the number of n = 150 pigs) was performed in a Danish herd from day 29 to 47 post weaning. Every third day all pigs were weighed, subjected to a clinical examination and faecal samples were obtained. All faecal samples were subjected to dry matter determination. Faecal samples obtained at day one, seven, 13 and 19 were subjected to quantitative PCR (qPCR) testing for L. intracellularis and PCV2.A longitudinal study  10 blood samples were collected and examined for L. intracellularis specific antibodies by ELISA as previously described[L. intracellularis and had not previously been subjected to antibiotic batch medication. At the first day of the study 10 faecal samples were obtained from pigs with watery faeces to verify an ongoing infection of L. intracellularis.The study was performed in one nursery herd selected at convenience. Criteria for selecting the herd was a history of infection with F systemto be freescribed. Based oOn day one of the study all 216 pigs in the selected room were subjected to a clinical examination and a faecal sample was obtained from each pig. Faecal consistency was clinically assessed by one observer using a clinical faecal consistency scale with four categories as previously described. FurtherDuring the study period each pig was subjected to a clinical examination, weighed (precision +/− 100 gram) and a faecal sample of minimum 2 gram faeces was obtained by digital manipulation of rectum on days one, four, seven, 10, 13, 16 and 19. On the remaining days pigs were subjected to a visual clinical examination. All examinations were performed by the same observer starting at the same time of day and the pens were examined in the same order. Pigs were re-examined later within the same day if less than 2 gram of faeces was obtained at the examination.Treatment of pigs was not allowed during the study period. However to apply to the Danish welfare legislation pigs were treated with antibiotics and excluded from the study if they were experiencing depression, dehydration, bloody diarrhoea, necrotic material in faeces and/or poor body condition. Treated pigs were removed form the pens at the day of treatment.At the end of each examination day the obtained faecal samples were transported by car to the National Veterinary Institute and stored at 4°C. Processing of faecal samples was performed the following day. Blinding was applied in relation to all laboratory examinations.All faecal samples were subjected to faecal dry matter determination. The individual faecal samples were homogenized with a spoon. Faecal dry matter content was determined for minimum one gram of faeces by drying to constant weight using a microwave oven as previously described.L. intracellularis/PCV2 positive sample and one L. intracellularis/PCV2 negative sample were included. The L. intracellularis/PCV2 negative sample contained DNA from E. coli as determined by qPCR specific for the two included E. coli strains (data not shown). The L. intracellularis/PCV2 positive extraction control contained DNA from L. intracellularis and PCV2 as determined from each specific qPCR (data not shown).A suspension of 10% faeces in phosphate buffered saline (PBS) was prepared from each faecal sample. The individual faecal samples were homogenized with a plastic spoon and 0.1 gram of faeces was suspended in 0.9 gram of PBS. The faeces suspension was stored at −80°C until further processing. Total DNA was extracted from the 10% faecal suspensions by using QIAsymphony extraction robot and QIAsymphony Virus/Bacteria Mini Kit (QIAGEN). The protocol was Pathogen Complex 200, the processed sample volume was 200 μl and elution was done in 110 μl. Prior to DNA extraction the 10% faecal suspensions were pre-treated by bead beating in Tissuelyser (QIAGEN) for 20 s and 15 Hz at room temperature with 5 mm stainless steel beads (QIAGEN). The suspensions were centrifuged for 90 s at 6,600 g  and the supernatant was transferred to the QIAsymphony robot. In each DNA extraction experiment one L. intracellularis were as previously described[L. intracellularis a standard curve was made from 10% qPCR negative pig faeces and spiked with a 10-fold dilution of the reference strain L. intracellularis (ID # 15540)[L. intracellularis specific qPCR was 4.3-8.3 log10 bacteria/g faeces. The limit of detection was 3.3 log10 bacteria/g faeces. All qPCR conditions for PCV2 were as previously described for the PriProET assay[10 genome copies/g faeces. The limit of detection was 4.0 log10 genome copies/g faeces.All qPCR conditions for escribed. For quaris ID # 540[L. iL. intracellularis bacteria and PCV2 copies in faeces) were associated to the ADG for the previous seven days in the individual pigs. For each pig three ADG intervals were calculated:For each of the observation days seven, 13 and 19 it was investigated whether the laboratory results  = (weight on day seven - weight on day one)/62 ADG day seven to 13 (kg/day) = (weight on day 13 - weight on day seven)/63 ADG day 13 to 19 (kg/day) = (weight on day 19 - weight on day 13)/6L. intracellularis bacteria and PCV2 copies were log10 transformed before the statistical analysis. Further, the partly ordinal/partly continuous scales for both qPCR tests were taken into consideration. In order to use the continuous part of the qPCR scales a number of new variables were generated. First an ordinal variable for L. intracellularis was generated by categorising the qPCR results into zero = L. intracellularis not detected, one = L. intracellularis qPCR positive below the dynamic range (4.3 log10 bacteria/g faeces), two = number of L. intracellularis within dynamic range and three = number of L. intracellularis above the dynamic range (8.3 log10 bacteria/g faeces). A new continuous variable was generated. The values for this variable were equal to the determined number of L. intracellularis in log10 bacteria/g faeces for faecal samples containing L. intracellularis within the dynamic range. For samples below or above the dynamic range this variable was set to zero. Both new variables were included in the same model in the statistical analysis. A similar strategy was used in relation to analysis of PCV2.The laboratory results for faecal samples obtained on day seven were tested for association to ADG day one to seven, laboratory results for faecal samples obtained on day 13 were tested for association to ADG day seven to 13 and laboratory results for faecal samples obtained on day 19 were tested for association to ADG day 13 to 19. Therefore each pig provided a maximum of three observations  to the statistical analysis. The number of 10L. intracellularis bacteria/g faeces and log10 PCV2 copies/g faeces were investigated using a multilevel mixed-effects linear model with observation day included as random slopes at pig and pen levels to account for the repeated measures on the individual pigs[L. intracellularis bacteria and the two variables containing the numbers of PCV2 copies were included as independent variables. Gender and start-weight (weight on day one of the study) were included as co-variables. The initial model was reduced by stepwise backward selection using a P-value = 0.05 as the selection criteria. For the reduced model interactions between main effects were investigated. Further, interactions between L. intracellularis and PCV2 were investigated. Interactions with a P-value below 0.05 were included in the final model. All analyses were performed using the xtmixed command in STATA IC version 11.Association between ADG, percent faecal dry matter, logual pigs. The mulAt day one 66 of the 216 pigs in the selected room were excluded because of diarrhoea or other clinical signs. Therefore a total of 150 clinical healthy pigs 29 days post weaning were included in the beginning of the study. The pigs were housed in 11 pens, 74 females and 76 males, mean weight = 16.0 kg (standard deviation = 2.55 kg). During the study 10 pigs were excluded because of treatment. Two pigs had to be euthanased. One of those pigs had previously been treated while the other one was euthanased at the end of the study.L. intracellularis bacteria and PCV2 genome copies in faeces for each observation day are displayed in TableL. intracellularis bacteria and ADG for the previous seven days prior to collection of the faecal samples is displayed in FigureData was missing randomly on a few other observation days providing a total of 428 observations for the statistical analysis. ADG, faecal dry matter content, numbers of L. intracellularis log10 bacteria/g faeces was significantly associated to decreasing ADG (P < 0.001). This association was dependent on the faecal dry matter content as a significant interaction between the number of L. intracellularis log10 bacteria in faeces and percent faecal dry matter was observed (P < 0.01). The negative association between the number of L. intracellularis and ADG was increasing with decreasing faecal dry matter content, FigureL. intracellularis was not significantly different (P = 0.26) from ADG in pigs with low numbers of L. intracellularis (below dynamic range) in faeces. Increasing start-weight was significantly associated to increasing ADG (P < 0.001) and ADG was significantly different between the three ADG intervals (P < 0.001). Gender (P = 0.29) and number of PCV2 log10 copies/g faeces was not significantly associated to ADG (P > 0.5). Expected ADG  obtained from the multilevel mixed-effects linear model for a number of fictive qPCR and faecal dry matter measurements are displayed in Figure The result of the statistical analysis (multilevel mixed-effects linear model) is displayed in Tablent P = 0.6 from ADA SPF herd was selected to avoid any confounding effect of other major pig infections in relation to ADG. Further, a short observation period was selected to minimize the risk of time varying co-variables that could influence ADG during the study.L. intracellularis bacteria in faeces were unaffected compared to L. intracellularis (qPCR) negative pigs. This is in accordance to a previous report that pigs under some circumstances can excrete detectable levels of L. intracellularis in faeces without a reduction in ADG[L. intracellularis bacteria even in pigs without diarrhoea  supports previous reports of a substantial negative impact of subclinical infections with L. intracellularis[L. intracellularis bacteria was dependent on the faecal dry matter content. Similar, decreasing faecal dry matter was associated to decreasing ADG but was dependent on numbers of L. intracellularis bacteria. Therefore the effect of L. intracellularis or faecal dry matter on ADG can not be assessed in practice without taking both into consideration. With decreasing faecal dry matter the negative association between ADG and L. intracellularis was increasing. Interesting in pigs with higher faecal dry matter (<20%) the association between ADG and L. intracellularis was minimal. Further, it seems that in pigs with faecal dry matter above 25%, infection with L. intracellularis has no negative effect on ADG. This aspect should be further investigated as it has implication for interpretation of L. intracellularis PCR test results in practice. A possible biological explanation for this observation could be that pigs with lower faecal dry matter are most affected by the L. intracellularis infection and will be more affected by increasing infection severity (as measured by L. intracellularis bacteria in faeces).The ADG in pigs with a low number of n in ADG. The obsellularis. HoweverVaccination against PCV2 has been reported to prevent PCVAD and thereby reduce the negative impact on ADG and the L. intracellularis , total PCV2  and average faecal dry matter . These strategies all provided similar results and identical conclusions in relation to the association between ADG, faecal dry matter content, number of L. intracellularis bacteria and PCV2 copies in faeces. The retrospective design was selected for the paper, because these results were considered the most useful in veterinary practice. Pig veterinarians are often consulted by farmers for investigation of groups of pigs with diarrhoea and/or reduced performance. The decreasing ADG observed for increasing numbers of L. intracellularis bacteria in faeces suggest that determination of the number of L. intracellularis bacteria in faeces can be applied for economic assessments of infections with L. intracellularis. Further studies are needed to investigate differences between herds and obtain more accurate estimates for the observed associations.In the statistical analysis a retrospective design was applied using the qPCR results and faecal dry matter content at the same observation day to estimate the ADG in the week prior to collection of the examined faecal sample. Other analytic designs were also applied to the same data (data not shown), including testing the association between ADG (measured from day one to 19), total L. intracellularis bacteria in faeces was associated with decreasing ADG in the week prior to collection of the examined faecal sample. The magnitude of the association was increasing with decreasing dry matter content of the examined faecal sample. Therefore faecal dry matter must be taken into consideration for interpretation of the effect of L. intracellularis. The number of PCV2 genome copies in faeces was not associated to ADG. No interaction between PCV2 and L. intracellularis was observed.An increasing number of The authors declare that they have no competing interests.All authors conceived and designed the study. KSP and RS performed all clinical herd investigations. ØA, MS, CH and LEL performed all PCR experiments. KSP conducted the statistical analyses. All authors participated in drafting the manuscript. All authors have read and approved the final manuscript.


The early identification of pre-lingual deafness is necessary to minimize the consequences of hearing impairment on the future communication skills of a baby. According to the most recent international guidelines the deafness diagnosis must occur before the age of three months and the prosthetic-rehabilitative treatment with a traditional hearing aid should start within the first six months. When a Cochlear implant becomes necessary, the treatment should start between the age of 12 months and 18 months. The only way to diagnose the problem early is the implementation of universal neonatal audiological screening programs. Transient evoked otoacoustic emissions (TEOAE) is the most adequate test because it's accurate, economic and of simple execution. Automatic auditory brainstem response (AABR) is necessary to identify patients with auditory neuropathy but it is also important to reduce the number of false-positives.The 20-30% of infant hearing impairment is represented by progressive or late-onset hearing loss (HL) so it's also necessary to establish an audiological follow up program, especially in infants at risk.From November 2005 all neonates born in the University hospital of Pisa undergo newborn hearing screening. From 2008 the screening program follows the guidelines for the execution of the audiological screening in Tuscany which have been formulated by our group according to the 2007 JCIH Position Statement and adaptated to our regional reality by a multidisciplinary effort. From November 2005 to April 2009 8113 neonates born in the Neonatal Unit of Santa Chiara Hospital (Pisa) have undergone newborn hearing screening. 7621 neonates (93.9%) without risk factors executed only the TEOAE test. 492 (6.1%) neonates had audiological risk factors and thus underwent TEOAE and AABR. 84 patients  failed both TEOAE and AABR tests. 78 of them underwent further investigations. 44 patients resulted falsepositives . 34 neonates  had a final diagnosis of hearing impairment. 8 patients (0.99 ‰) had unilateral hearing loss (HL). 26 patients  had bilateral hearing impairment.In our screening program the percentage of false-positives was quite low (0.54%) while the incidence of bilateral HL (3.2 ‰) is a little higher than that found in literature reports. In most of our patients premature birth or neonatal suffering represent the main cause of HL. The identification and the early diagnosis of pre-lingual deafness is necessary to prevent or minimize the serious consequences of hearing impairment on language development and on the future communication skills of a baby -7.According to the most recent international guidelines the deafness diagnosis must occur before the age of three months and the prosthetic-rehabilitative treatment with a traditional hearing aid should start within the first six months. When a Cochlear implant becomes necessary, the treatment should start between the age of 12 months and 18 months . Pre-linThe aim of such programs is to identify hearing impairments present at birth, overall medium and severe  -21,6,7.The most important international guidelines suggest the execution of a universal screening program and hence screening tests should be done on all neonates and not only on those presenting increased risk factors -7,22, asBoel test. Boel test is difficult to execute (it requires specific expertise) and it successfully identifies only less than half of babies with a hearing impairment. That explains why, in the absence of a screening program, the average delay in the diagnosis ranges between 18 months and 24 months. Such delay might cause a decreased effectiveness of the rehabilitation therapy and irreparable consequences for the patient [Actually in Italy neonates without risk factors are tested at around 8 months of age with the use of the  patient -14,29,30The aim of the universal screening is hence to identify as early as possible the highest number of infants with permanent bilateral hearing impairments ,15,22,31Recent screening methodologies (TEOAE and AABR) are completely risk-free and extremely accurate ,7,32.When the latest technology is used in conjunction with specific operator training, the accuracy of screening programs can be close to 100% with a specificity of about 97-98%. That means that virtually all neonates with a hearing impairment greater than 40dB will be identified, while about 2-3% of the babies diagnosed will be false positives. The false positives are later re-examined and a large part of them subsequently presents normal hearing function -35.In the last 10 years a new clinical/audiological entity has been defined (but not completely understood): the auditory neuropathy. Auditory neuropathy is characterised by normal otoacoustic emissions and altered auditory brainstem response, due to damage either to the inner hair cells, damage to the acoustic nerve fibres or to the synaptic junction between them and inner hair cells ,36-38.Neonates affected by auditory neuropathy can be diagnosed only by executing the ABR test  in conjunction with otoacoustic emissions. A TEOAE test in this cases would lead to a falsely negative outcome. For these reasons it is necessary to test neonates with auditory neuropathy risk factors with both TEOAE and AABR tests ,36.In the last few years there has been an increased focus on late onset hearing loss. Progressive or late onset deafness can have different causes  and represents a relatively large percentage (20-30%) of hearing loss in children, even if reliable statistics are not available internationally . Babies The Joint Committee on Infant Hearing (JCIH) in the 2007 Position Statement has identified the issue of late onset hearing loss and has defined the risks factors that requires an audiological follow-up during the first years of life [Regione Toscana) has made compulsory the execution of the neonatal audiological screening program in all the birth centers of the region.With the Regional Decree n.365 of May 21st 2007, the local regional government of Tuscany  in June 2008 and are today a reference for all the hospitals and operators  executing screening programs in Tuscany. Our guidelines are based on the ones created by the JCIH in 2007, but have been modified by a multidisciplinary effort and adapted to our regional reality. Follows a brief example showing the methods of screening execution in Tuscany.We have subsequently written the "Guidelines for the execution of the neonatal audiological screening" together with the Department of Audiology of the Careggi Hospital (headed by Prof P Pagnini) and with the Audiology Department of the University of Siena (headed by Prof W Livi). The guidelines were approved by the The screenings can be executed in three different types of hospitals which are distinguished by their level. A centre is assigned a level depending on the type of instruments and diagnostic equipment available.Level I facilities: these facilities can only execute TEOAE tests with latest available diagnostic instruments. The test is executed by audiometrists, audiologists, paediatricians, paediatric nurses or otorhinolaryngologists after a specific training.Level II facilities: these facilities have the latest available instruments necessary to execute TEOAE and AABR tests. Also in this case the test is executed by trained audiometrists, audiologists, paediatricians, paediatric nurses or otorhinolaryngologists.Level III facilities : these hospitals have the latest equipment for the execution of TEOAE, AABR and they are also capable of executing clinical ABR, clinical TEOAE and DPOAE (distortion produced otoacoustic emissions), impedence audiometry and infant audiometric testing.All these diagnostic tests are executed by personnel with a specific expertise in this field. Level III facilities can perform an early diagnosis, an investigation on the causes of the hearing impairment and the initiation of a prosthetic/rehabilitative treatment. Each facility should at first classify neonates depending on whether they present auditory neuropathy risk factors or no because well babies and neonates at risk should perform different kind of screening test battery.In all cases the screening must be executed before discharging the neonate from hospital and positive tests might be repeated within two weeks after birth. Each local health authority should nominate a person in charge of the screening procedures in each facility. This person might be a paediatrician, neonatologist, audiologist or an otorhinolaryngologist and he/she should involve the audiometrists and the paediatric nurses in the process. In general is advised that the screening should be performed only by dedicated personnel with a specific training and not by generic health operators. Especially in facilities of Level II and III, the screening should be performed by audiometrists and there should be a close collaboration between the Neonatal Units and the Audiology Units.The Clinical Physiology Institute of the CNR  is developing a database that will be used by all regional facilities to file and retrieve neonatal audiological screening data. Such data will then be made available online. This will allow a monitoring on the effectiveness of the screening program .All these entities will need to establish the details of the screening execution for the babies born in private clinics, babies born at home, in other Italian regions or other countries. All these babies will need to be screened within a month from birth or within a month from the moment they are assigned to a family paediatrician.pass) for both ears the audiological screening is considered successfully completed. If instead the test outcome is positive (refer) for one or both ears then the TEOAE test should be repeated before the infant is discharged from hospital. If this second test still produces a positive result the infant should undergo a AABR test. Level I facilities should send these neonates to level II or level III facilities where AABR can be performed. AABR should be executed within 30 days from birth. If the complete test (TEOAE+AABR) outcome is positive, infants should undergo further tests in a level III facility, within 90 days from birth, such as clinical ABR, clinical TEOAE etc. Level III facilities should then start the investigation on the causes of the hearing impairment and should initiate a prosthetic/rehabilitative treatment within 6 months from birth.Dedicated personnel  should execute the TEOAE test 24 hours after birth. Tests should be performed in silent rooms while the neonates are asleep or when they are most quiet (for example after feeding). If the test outcome is negative  should be immediately investigated in all infants that result positive at the first level audiological screening (TEOAE), researching CMV DNA in urine.Auditory neuropathy risk factors are:- neonatal intensive care of more than 5 days or any of the following regardless of length of stay - family history of permanent childhood hearing loss- family history of neurodegenerative disorders, such as Hunter syndrome, or sensory motor neuropathies, such as Friedreich ataxia and Charcot-Marie-Tooth syndrome.Neonates with auditory neuropathy risk factors should undergo both automatic TEOAE and AABR test before the discharge from the hospital (or within 30 days from birth if neonates are born in a level I facility).In premature neonates audiological screening should be executed after 35 weeks of gestational age. If the test produces a positive result it should be repeated before discharge or within two weeks. In all infants where audiological tests produce a definitely positive result a Cytomegalovirus (CMV) infection should be investigated by detection of CMV DNA in urine. If the second test still produces a positive result the infant should be sent to level III ORL Department to perform further tests within 3 months from birth.Before the infant is discharged from the hospital the person responsible for the screening should write the screening results on the paediatric booklet , on the screening's results register and on the online database. The person responsible should specify which kind of test  the neonate has undergone and what result (positive or negative) the test has produced. The screening manager should also specify the timing for further audiological investigation depending on neonate's risk factors. Parents should be given adequate information on congenital hearing impairment if their neonate has failed newborn hearing screening. Infants of a few months of age re-admitted to hospital within because of hyperbilirubinemia requiring exsanguino-transfusion or for a sepsis diagnosed with positive culture should repeat TEOAE and AABR test regardless to their neonatal screening result.Progressive and late-onset deafness account for about 20-30% of infantile hearing loss. These patients can develop later hearing impairment even if the neonatal audiological screening was successfully completed. The only way to identify them is to keep monitoring infants with risk factors for progressive or late-onset hearing impairment throughout their childhood regardless to their neonatal audiological screening result. Patients with the following risk factors should undergo at least one audiological evaluation between 24 and 30 months of age:- neonatal intensive care of more than 5 days or any of the following regardless of length of stay ,- In-utero infections, such as herpes, rubella, syphilis, and toxoplasmosis- Craniofacial anomalies, including those that involve the pinna, ear canal, ear tags, ear pits, and temporal bone anomalies- Physical findings, such as white forelock, that are associated with a syndrome known to include a sensorineural or permanent conductive hearing loss.Boel test should be performed by the family paediatrician as usual.Patients with the risk factors listed below should undergo audiological evaluation every 6-12 months till 3 years of age and then every 12 months till 6 years of age.- In-utero CMV infection- family history of permanent childhood hearing loss- Syndromes associated with hearing loss or progressive or late-onset hearing loss, such as neurofibromatosis, osteopetrosis, and Usher syndrome; other frequently identified syndromes include Waardenburg, Alport, Pendred, and Jervell and Lange-Nielson.- Neurodegenerative disorders, such as Hunter syndrome, or sensory motor neuropathies, such as Friedreich ataxia and Charcot-Marie-Tooth syndrome- Culture-positive postnatal infections associated with sensorineural hearing loss, including confirmed bacterial and viral  meningitis- Head trauma, especially basal skull/temporal bone fracture that requires hospitalization- Chemotherapy or ototoxic drugs- Caregivers concern regarding hearing, speech, language, or developmental delayBoel test should be performed by the family paediatrician as usual.Family paediatricians should be part of his screening program by investigating auditory function in infants and children with and without risk factors during routine health checks at 3,6,8,12,18 months and at 3 and 6 years.From 1993 to the 20th of november 2005 neonates with audiological risk factors have been evaluated with clinical ABR.refer) can be performed thanks to the collaboration between the U.O. Neonatologia, AOUP and the Otology and Cochlear Implant Centre of the University hospital of Pisa. TEOAE and AABR tests are executed with the device Accuscreen Pro-GN Otometrics- by an audiometrist with the collaboration of dedicated medical personnel. Tests are performed following the guidelines described above. TEOAE are executed on all newborns. The test is repeated before discharge when the first examination produces a positive result. If the second test still produces a positive (refer) result neonates undergo AABR test.From the 20th of November 2005 all neonates born in the University hospital of Pisa (which represents the reference facility of the North-West Tuscany Area) undergo newborn hearing screening. They execute the test (TEOAE or AABR depending on the presence or absence of auditory risk factors) before the discharge from hospital or within 2 weeks from birth. This test, and further investigations in newborns presenting positive test outcome  criteria . If hearing loss (HL) is confirmed further biochemical and instrumental tests are carried out. When the cause of HL can't be detected, DNA is extracted from the Guthrie card which had been stored in the first days from birth to search for CMV genome. DNA is extracted with DNA blood minikit  and visualized on agarosio gel.AABR are immediately executed in neonates with audiological risk factors (risk factors are based on those reported on the JCIH Position Statement 2000 and 2007). When AABR produces a positive result further audiological tests are executed. Hearing impairment severity degree is classified according to the BIAP  have undergone newborn hearing screening (Table refer). 492 (6.1%) of them had audiological risk factors and thus underwent TEOAE + AABR test. 84 patients , among which 39 with risk factors and 45 without risk factors failed both first (TEOAE) and second level (AABR) tests. These 84 infants underwent audiological evaluation at Otology and Cochlear Implant Centre of the University hospital of Pisa. Only 6 of them (0.07% of the 8.113 neonates) (two with risk factors and four without) didn't execute further examinations because parents didn't agree to further tests (2 cases); because the hospital was unable to get in touch with the parents (2 cases); because of life-threatening conditions (1 case); or because they died (1 case). 78 infants underwent clinical ABR and further tests. 24 resulted pass at the first test. 15 failed the first test but resulted pass at the second ABR test. In other 5 patients the third examination with AABR gave a pass result. Finally 44 patients resulted false-positives .7621 neonates (93.9%) without risk factors executed only the TEOAE test  had a final diagnosis of hearing impairment. 8 patients (0.99‰) (only one had risk factors) had unilateral hearing loss . 26 patients  had a final diagnosis of bilateral hearing impairment : 5 mild , 11 medium  and 10  severe/profound HL. 21 of them  had risk factors . In one patient auditory neuropathy was diagnosed.CHD7 gene; 1 case of Diamond Blackfan Anemia; 1 case of 5q11.2-q13 duplication; 1 case of malformative syndrome in a female with q11 deletion plus partial trisomy of chromosome 6); 3 patients had congenital CMV infections (one of them was positive for CMV and heterozygous mutation R127H on GJB2 gene).Among the 26 bilaterally hearing impaired patients: 12 cases were prematurely born or had suffered from respiratory problems ; in 2 patients connexine 26 gene mutations were found ; 1 patient had family history of infantile deafness; 4 patients had syndromes related to hearing impairment . The 34.1% of false positive cases are patients with risk factors.pass at the first ABR, 15 at the 2nd, 5 at the 3rd) the hearing threshold of ABR improved after the repetition of the test, probably in relation to a maturation of the auditory system. Some patients with hearing threshold lower than 40 dB at the first ABR test gained a normal hearing threshold over a follow-up of six months. Physiological immaturity of central auditory nervous system and physiological narrowness of auditory canal can thus explain most of false positive results if audiological tests are performed in the first few days after birth.We also underscore the importance of a follow-up in newborn who have failed the newborn hearing screening. In some cases  is a little higher than that found in international and national literature.Perhaps this difference is due to the fact that our data have been collected in a level III facility with a neonatal intensive care unit so many neonates which have been tested have undergone neonatal intensive care for more than 5 days, they have required assisted ventilation, they have been exposed to ototoxic medications or they have gained high levels of hyperbilirubinemia .It's important to identify the etiology of HL so that the treatment can be more individualized. In our study we could not identify the causes of HL in only 4 patients. In 12 neonates  the main cause is premature birth or neonatal respiratory distress.The implementation of this screening program has required a considerable organizational effort and the employment of dedicated personnel which has absorbed resources otherwise available to other audiological activities. Four patients referred to the screening program did not turn up for the tests despite repeated invitations from the hospital. This situation occurred in 4.76% of the referred neonates - 0.05% of all the screened patients. It's also essential, in our opinion, family pediatricians to be involved in a follow-up program in order to detect children with progressive or lateonset HL.The authors declare that they have no competing interests.PG and SB conceived the study, participated in its design and coordination and drafted the manuscript. AL participated in the design of the study and performed audiological screening (TEOAE and AABR). FF participated in the design of the study, performed further audiological investigation and drafted the manuscript. SL participated in the design of the study, performed audiological screening and drafted the manuscript. AB and AB participated in drafting the manuscript and in the analysis of the results.All authors have read and approved the final manuscript.