Introduction. Vestibular schwannomas (VS) are benign tumours of the vestibular nerve and can lead to hearing loss, tinnitus, vertigo, facial palsy, and brainstem compression. Audiovestibular diagnostic tests are essential for detection and treatment planning. Methods. Medline was used to perform a systematic literature review with regard to how audiovestibular test parameters correlate with symptoms, tumour size, and tumour location. Results. The auditory brainstem response can be used to diagnose retrocochlear lesions caused by VS. Since hearing loss correlates poorly with tumour size, a retrocochlear lesion is probably not the only cause for hearing loss. Also cochlear mechanisms seem to play a role. This can be revealed by abnormal otoacoustic emissions, despite normal ABR and new MRI techniques which have demonstrated endolymphatic hydrops of the inner ear. Caloric and head impulse tests show frequency specific dynamics and vestibular evoked myogenic potentials may help to identify the location of the tumour regarding the involved nerve parts. Conclusion. In order to preserve audiovestibular function in VS, it is important to stop the growth of the tumour and to avoid degenerative changes in the inner ear. A detailed neurotological workup helps to diagnose VS of all sizes and can also provide useful prognostic information. Vestibular schwannoma (VS) can lead to sensorineural hearing loss, tinnitus, and vertigo. Patients with asymmetric hearing loss, unilateral tinnitus, or vertigo should be evaluated for the presence of a VS.VS are benign tumours arising from the sheath of the vestibular nerve. They cause dysfunction of the nerves by compressing the nerve fibre and obstructing the blood supply to the nerves , 2. VS mThe estimated annual incidence of these tumours ranges from approximately 0.6 to 1.9 per 100,000 .Currently there exist several classifications of VS, based on the tumour size and location. Audiovestibular function tests, such as pure tone audiometry (PTA), otoacoustic emissions (OAEs), auditory brainstem response (ABR), caloric irrigation, head impulse test (HIT), and vestibular evoked myogenic potentials (cVEMPs and oVEMPs) are important for initial diagnosis as well as subsequent monitoring of disease progression.The current gold standard of diagnosis is MR imaging, but because MRI scans are expensive, they are not universally utilized to screen all patients with audiovestibular symptoms . Thus, VFurthermore, precise preoperative audiovestibular assessment can have implications on the decision of the therapeutic approach such as wait and scan, radiotherapy, or microsurgery.English and German articles published between 1972 and 2016 and available on Medline were reviewed in order to produce a summary of the current knowledge with regard to audiovestibular functional changes in patients with VS as diagnosed by MR imaging.Sudden sensorineural hearing loss may be an initial symptom of a VS ; therefoIt has been shown that VS has been diagnosed in 0.8–47.5% of patients with sudden sensorineural hearing loss , 7.To investigate predictive factors or symptoms of future hearing loss Jethanamest et al. retrospectively reviewed patients between 2002 and 2013 who chose observation for their initial management. The estimated median time to loss of serviceable hearing was 6.3 years. No symptoms or factors were identified to be predictive of future hearing loss. However, symptoms of disequilibrium or imbalance appeared to be associated with an increased risk of subsequent tumour growth .p < 0.001) but not by tumour size. At follow-up examinations after treatment with microsurgery and radiotherapy, hearing was significantly worse in those patients with a large VS. Age was not a significant determinant for loss of hearing on the follow-up visits (p < 0.08) [Wagner et al. found that the degree of hearing loss before treatment is significantly influenced by the age of the patient ( < 0.08) .Another study noted that 45% of those patients who initially had a hearing ability of AAO class A on the tumour affected side lost class A hearing after 5 years .Concerning speech discrimination, Stangerup et al. demonstrated during a 33-year period that 59% of the patients with a speech discrimination better than 70% preserved good hearing after a mean of 4.7 years of observation. 69% of the patients with a speech discrimination score of 100% at diagnosis maintained good hearing after more than 10 years. Of the patients with only a small discrimination loss at diagnosis, 38% maintained good hearing .Tumour growth less than or equal to 2.5 millimeters per year has been shown to lead to higher rates of hearing preservation. For patients with a small tumour and normal speech discrimination, the main indication for active treatment should be the established tumour growth , 12.Day et al. found in a total of 44 patients that normal hearing or low-frequency hearing loss was correlated with small tumour size. Those with mid- or high-frequency hearing loss had a medium-sized tumour, while those with global frequency hearing loss or total deafness had tumour size larger than 2.5 cm. The authors explained the hearing loss by tumour compression of the cochlear nerve, since most VS originate from the vestibular nerve . This asp < 0.01) [Concerning primary inner ear schwannomas (PIES), that is, schwannomas arising from the labyrinth, a systematic review of 243 patients diagnosed from 1933 to 2011 showed that unilateral hearing loss was the most frequent presenting symptom (99%). Vertigo and abnormal balance were more common among tumours involving the vestibular system ( < 0.01) . Positio < 0.01) . Especia < 0.01) .Overall, there is limited data on the long-term auditory symptoms in patients with sporadic small- and medium-sized VS. The initial treatment strategy for VS is being discussed controversially. The overall prognosis for hearing in sporadic VS is poor regardless of treatment strategy. Good baseline hearing proved to be a strong predictor for maintained serviceable hearing. Observation was associated with the highest rate of hearing preservation , althougAfter audiometry, the next diagnostic test of choice for patients with clinical suspicion of a VS  was the ABR. However, with improvements in MRI diagnostics in the late 1980s, the usefulness of the ABR in the diagnostic workup of VS has diminished and MRI is nowadays widely considered to be the gold standard for diagnosing retrocochlear pathology.Currently, the role of the ABR remains controversial. The criteria for assessing abnormal responses on ABR tests have varied considerably among studies. Some authors argue that the ABR has no place in today's management and that MRI should be the initial screening test when there is a suspicion of VS . Other aThe ABR has a high dependency on the tumour size and the diagnostic sensitivity is especially low for smaller tumours .This was also shown in a study by Koos et al. in which multiple online databases were assessed in order to evaluate the usefulness of ABR tests in 3314 patients from 1978 to 2009. The inclusion criterion was the presence of a surgically or radiographically confirmed VS. The pooled sensitivity of ABR was 93.4%, 85.8% for tumours < 1 cm, and 95.6% for tumours > 1 cm. Moreover, the sensitivity of ABR was higher for extracanalicular than for intracanalicular tumours. The authors concluded that for patients with lower clinical suspicion of a VS an ABR can still provide valuable additional information on which the decision to obtain an MRI can be based.However, a small number of tumours with audiometrically documented hearing loss demonstrate a normal ABR , which sOAEs are active mechanical responses from the cochlea which provide information about the integrity of the preneural cochlear receptor mechanisms. Clinical experience has shown that OAEs are often absent or compromised in VS. In order to objectify the effects of retrocochlear disease on distortion-product otoacoustic emissions (DPOAEs) Telishi et al. classified the DPOAE patterns as cochlear or noncochlear. In a large series of patients with unilateral VS they showed that the majority of ears with tumours demonstrated cochlear (57%), rather than noncochlear (41%) patterns of DPOAEs .The occurrence of cochlear pathology has implications regarding the accuracy of OAEs as a diagnostic tool for VS. If one assumes that VS affect the retrocochlear function  and have little effect on the cochlea itself then VS patients should have preserved OAEs. However, it seems that amplitudes of DPOAEs begin to decrease already at the early stages of hearing loss in VS patients. Comparing a group of VS patients with normal/symmetrical hearing and a group of VS patients with mild hearing loss (threshold at any tested frequency better than 45 dB) on the tumour ear side, Gouveris et al. showed that DPOAE amplitudes do not differ strongly between the ears in VS patients with normal/symmetrical hearing. But DPOAEs are decreased compared with the nontumour ear in VS patients with even mild hearing loss. The tumour size did not differ significantly between these two groups, which suggests a cochlear origin of early hearing loss in these patients. The authors conclude that DPOAEs may be used in a clinical setting to monitor progression of cochlear damage at the early stages of hearing impairment in VS patients .Vertigo has been reported to be one of the risk factors for the growth of VS . TherefoWith the caloric test it is possible to unilaterally stimulate the horizontal semicircular canal, which is innervated by the superior part of the vestibular nerve. Thus, one might think that the caloric response is only significant when the superior branch of the vestibular nerve is affected by the VS. Borgmann et al. tested whether ENG results could predict the nerve of origin before surgery. They defined pathologic caloric test findings as an indicator for superior vestibular nerve schwannomas (SVN) involvement and a normal caloric response as a sign of inferior vestibular nerve schwannomas (IVN) origin. As a result, the nerve of origin could be predicted in 90 of 111 patients (81%), which means that pathologic results in preoperative ENG were significantly more frequent in patients with SVN schwannomas. They concluded that the caloric test helps to predict the nerve of origin of a VS and can be used indirectly as a prognostic factor for hearing preservation because hearing loss due to surgery was significantly lower in patients with tumours of the SVN .In contrast Ushio et al. demonstrated in a study of 109 VS patients that the percentage of abnormal responses in caloric tests was not different between patients with SVN and those with IVN . These rTringali et al. observed a good correlation between caloric weakness and tumour size when they tested the preoperative response of 629 tumour patients. In the group of patients with UW > 70%, who had a larger tumour size, postoperative facial palsy was more frequent. Postoperative hearing preservation was more frequently observed in the “normal group” with a UW < 20%. They concluded that a normal caloric response can be a good predictive factor for hearing preservation and normal postoperative facial function .In a prospective pilot study on patients with VS, Wagner et al. compared groups with VS < 20 mm and ≥ 20 mm. In group 1, the median loss of vestibular function was +10.5%; in group 2 (with a tumour size ≥ 20 mm) a higher degree of loss of vestibular function (median UW 36%) was found. Treatment by micro- and radiosurgery caused a further decrease of vestibular function in both groups. So vestibular function clearly correlated with the size of the tumour and also with the method of treatment used. The majority of patients complained about vertigo before and after treatment .Ushio et al. also found that the mean tumour size in patients showing abnormal responses in caloric testing was larger than that in patients showing normal responses. This tendency was not observed for patients with VS within the internal acoustic canal . The samOn the contrary, the results of a study by Teggi et al. showed that vestibular function is influenced by intracanalicular length and diameter of the tumour, rather than by total tumour volume. The subgroup of 30 patients with a greater intracanalicular length of the tumour presented a higher value of UW than the subgroup with a smaller length [In summary, the available data suggest that the severity of the UW correlates with degree of infiltration of the cochleovestibular nerve by the tumour. This means that an abnormal caloric response is a prognostic factor for hearing preservation surgery .Both the caloric irrigation and the head impulse test (HIT) serve to evaluate the horizontal vestibuloocular reflex (VOR). The HIT applies high-acceleration small-amplitude head impulses around an earth-vertical axis while the patient is fixating a stationary target. A catch-up saccade is observed if the eye no longer compensates for the head movement and accordingly this test is classified as pathologic. The probability of a pathologic HIT generally increases with increasing UW of caloric examination. For patients with a UW within normal limits, a pathological HIT is very unlikely .At present the HIT is the only bedside examination that enables the identification of the side of a unilateral hypofunction of the peripheral vestibular system.However, a VOR deficit may not be diagnosed because corrective saccades cannot always be detected by simple observation. Besides the manual HIT as described by Halmagyi et al. , which c2013 Blödow et al. examined 142 patients with acute or chronic vestibular syndrome and found that 47.6% had a pathological vHIT whereas 52.4% had a normal test result. Covert catch-up saccades could be demonstrated in 13.7% whereas in 86.3% overt catch-up saccades alone or in combination with covert catch-up saccades were found. The authors concluded that the vHIT is superior to the clinical HIT because in approximately one in eight cases with acute or chronic peripheral vestibular syndrome a covert catch-up saccade can be detected, which would otherwise have been undetectable by the clinical HIT .The same group recently examined 46 patients with VS and showed that caloric irrigation exhibits a higher sensitivity than HIT (72% versus 41%) and both tests show only a moderate correlation. Tumour size and hearing level were significantly correlated with caloric abnormalities but not with HIT findings .The finding of a selective impairment of the VOR in the low-frequency range  was also shown in a case of an intralabyrinthine schwannoma, whereas the high-frequency VOR (HIT) remains intact . This coHowever, neither study could find an explanation for the higher sensitivity of the caloric test. Both tests measure the VOR but at different temporal frequencies: the HIT with short head impulses tests high frequencies up to 5 Hz  and the Vestibular testing at different frequencies provides deeper insights into VOR function and can help in detecting cerebellopontine lesions. According to the current literature, both tests have to be considered complementary and are valuable for both diagnostic and therapeutic decisions.The HIT and the bithermal caloric irrigation are different in terms of not only temporal frequency, but also the method of stimulation. The HIT causes a physiologic endolymphatic flow as a consequence of rapid head impulses. In contrast, caloric irrigation induces endolymphatic flow due to a temperature gradient from one side of the canal to the other. Caloric irrigation additionally stimulates the inner ear in a non-gravity-dependent way .It still remains unclear whether the dissociation of the caloric test and the HIT in patients with VS is due to the varying temporal frequencies or the differing methods of stimulation. Of note, the predilection for caloric test abnormality is typical for patients with Menière's disease  and has been shown to correlate with the degree of endolymphatic hydrops herniation into the horizontal semicircular canal. Therefore, this feature might suggest endolymphatic hydrops in some of these patients.VEMPs provide information on otolith organ function. The conventional method for recording VEMPs involves measuring electromyographic activity from surface electrodes placed over the tonically activated sternocleidomastoid muscles. The cervical VEMP (cVEMP) is a manifestation of the vestibulocollic reflex . VEMPs cIn recent years the role of cVEMPs and oVEMPs in the assessment of patients with VS has gained increased attention. A number of studies have demonstrated that VEMPs have an important clinical value in the diagnosis of VS because sometimes an abnormal VEMP result may be the only sign of a unilateral VS  .Physiological and clinical studies have shown that cVEMPs to ACS mainly reflect the function of saccular afferents and the inferior vestibular pathway –44.On the contrary, clinical studies in patients with unilateral VS , 46 and Recently VEMPs have been applied especially for classifying VS according to the involved nerves prior to surgery or radiotherapy.Iwasaki et al. postulated that BCV oVEMPs mainly reflect the function of the SVN. They hypothesised that if the oVEMPs to BCV mainly reflect the function of the utricular afferents, the results should mostly coincide with those of the caloric test rather than with cVEMPs to ACS. Among ten patients with inconsistent results in these three vestibular tests, seven (70%) showed corresponding results between oVEMPs to BCV and the caloric test, whereas only one patient (10%) showed correspondence between oVEMPs to BCV and cVEMPs to ACS , thus suIn a bigger cohort of 45 patients with untreated unilateral VS, the results of oVEMPs to ACS had a significant correlation with those of oVEMPs to BCV. These findings support the hypothesis that oVEMPs in response to both ACS and BCV are predominantly mediated by the superior vestibular nerve and probably both reflect the function of the utricle .However, according to other studies the nerve of origin of tumours cannot be predicted based on VEMPs. In a study of 130 patients histologically diagnosed by surgery, VEMPs in patients with tumours arising from the SVN were not significantly different from those in patients with tumours of the IVN . Ushio eWith regard to tumour size, several authors describe that, in VS, ACS cVEMPs are absent or decreased in amplitude in up to 80% of cases –51. The Tumour compression of the brainstem and the vestibular spinal tract and compression of the myelin sheath of the IVN, resulting in demyelination of the nerve, were also correlated with cVEMP latencies , 46.However, comparing the tumour size within the internal acoustic canal, Ushio et al. could not observe a difference between patients showing abnormal VEMP responses and patients showing normal responses .In summary, there exists at the moment no consensus about the use of VEMPS in detecting a VS. So it seems currently not possible to make any diagnostic prognosis through VEMPS.Since it is not yet clear whether there are symptoms or parameters (such as tumour growth) which can be used to predict future hearing outcomes in VS patients, one might raise the question whether the hearing loss is caused by retrocochlear or cochlear mechanisms. Clinical and histological observations have suggested that the hearing loss in VS does not correlate with the size of the VS , 54, indRoosli et al. performed a detailed assessment of cochlear pathology in patients with VS and also found a lack of correlation between tumour size and hearing loss. In a retrospective analysis of temporal bone histopathology they found that neural mechanisms caused by compression of the cochlear nerve are not solely responsible for hearing loss in patients with VS. When compared to the unaffected ear, VS caused significantly more inner and outer hair cell loss and cochlear neuronal loss, precipitate in endolymph and perilymph, and increased pure tone average thresholds. Moreover, the tumour distance from the cochlea and the nerve of origin did not correlate with structural changes in the cochlea or the hearing threshold. Direct invasion of the modiolus or the cochlea by VS was not observed in any case .It is nowadays possible to demonstrate endolymphatic hydrops in vivo through MR imaging  and the Naganawa et al. were able to identify vestibular endolymphatic spaces on noncontrast-enhanced 3D FLAIR (fluid attenuated inversion recovery) MRI. By using intrinsic perilymph signal increase in some patients with VS, endolymphatic hydrops was seen in four out of thirteen patients. The signal increase is attributed to the increased protein concentration . In thisIt has been suggested that metabolic abnormalities caused by the tumour can have an important impact on the symptoms and that in this way abnormalities within the labyrinth may result in secondary injury to the organ of Corti. Thus, the progressive hearing loss could be caused by the release of toxins or potassium ions by the tumour. However, these metabolites have never been isolated or shown to be causative , 63.The alteration of inner ear fluid composition in VS cases seems to play an important role. Different possible causes for this phenomenon have been described including impaired labyrinthine blood flow , 65, whiFinally, a small subset of patients may experience a mixed hearing loss, due to an inner ear conductive loss thought to be caused by interference with the intracochlear perilymphatic fluid wave or dampening of stapes movement , 72. Interference of microcirculation by nerve compression. Release of toxins or potassium ions by the tumour. Degeneration of the organ of Corti and the Stria vascularis. Changes of inner ear homoeostasis. Endolymphatic hydrops.The different causes of labyrinthine damage by a VS are summarized as follows:In order to preserve the auditory and vestibular function in VS patients, it is important not only to stop the growth of the tumour but also to avoid degenerative changes in the inner ear. A detailed neurotological workup helps in the diagnosis of VS of all sizes and can also provide useful prognostic information. In particular, caloric irrigation and unilateral hearing loss in audiometry have to draw the attention to the existence of a VS.As false-negative results remain possible in small intrameatal tumours (1%)  MR imagiLoss of vestibular function usually occurs slowly and gradually, allowing an effective central compensation, so vertigo symptoms are often absent until the loss of function is severe.Moreover, extended clinical vestibular function tests including vHIT and VEMPs can determine the overall extent of vestibular function loss, especially because the traditional caloric irrigation can deliver normal results in VS patients.It has been shown that there is a significant degeneration of labyrinthine structures in ears with VS. Nevertheless, it is still unclear whether VS symptoms like hearing loss and vertigo are caused primarily by these labyrinthine mechanisms or by retrocochlear mechanisms.Traditionally, VS have often been regarded simply as retrocochlear lesions. However, otoacoustic emission recordings and, in recent years, the endolymphatic hydrops visualization clearly show significant secondary labyrinthine pathology in patients with VS. Further research, using this new diagnostic possibility and correlating the morphologic findings with audiovestibular function test results in patients with VS will likely provide new insights into the relative contributions of neural versus labyrinthine factors to the pathophysiology and symptoms of VS. Some symptoms may possibly arise from labyrinthine factors rather than from neural factors. Since the inner ear is better accessible to therapeutic interventions than the audiovestibular nerve, for example, by intratympanic application of drugs, new therapeutic possibilities for symptom control in VS patients may possibly arise in cases where the tumour is stable and does not require invasive interventions.


Hearing deficits in children are demonstrably negatively associated with language acquisition and cognition. Although universal neonatal hearing screening exists, it is not offered equally across Canada. Additionally, children emigrating from other countries are often not assessed. The objective of this study is to evaluate Kids2Hear, a free hearing screening program run by medical students at elementary schools, and to determine the rate of hearing deficits that were identified and referred for evaluation.Retrospective analysis of screening program data from 228 participants seen at three inner-city elementary schools over six months.In our sample, the mean age was 5.8 ± 1.0 years with 48 % males. Approximately 21 participants (9.3 %) were screened positive for a hearing deficit and required referral for supplementary audiological evaluation. About 44 participants (19.3 %) were referred to a family physician for otoscopic abnormalities. Females were significantly more likely to be identified for both hearing deficits and otoscopic abnormalities.Hearing deficits and otoscopic abnormalities are common among young children. Female children may be at higher risk for developing hearing issues or otoscopic abnormalities compared to males. Additional research is needed to determine the effectiveness of hearing screening programs. Failure to identify children with congenital or acquired hearing loss can result in lifelong consequences including deficits in speech and language acquisition, poor academic performance, poor cognition , 2, poorAlthough most children with congenital hearing loss are identified by newborn hearing screening, some acquired or progressive hearing deficits may not be apparent until later in childhood . AcquireKids2Hear is a primary preventative health initiative run by medical students at the University of Toronto . In this program, trained medical students screen kindergarten students for hearing deficits and otoscopic abnormalities under the supervision of licensed audiologists and otolaryngologists from the Hospital for Sick Children . This free program is geared towards inner-city school students that reside in lower socio-economic areas as well as schools where there are a large proportion of children who have recently immigrated to Canada. Children who are identified with a hearing deficit through audiometry are referred to a pediatric audiology and otolaryngology clinic while children with otoscopic abnormalities are referred to a local family physician.The goal of this study was to evaluate a free hearing screening program offered at elementary schools and determine the rates of hearing deficits that were identified and referred for medical evaluation.This study was granted program evaluation status by the University of Toronto Research Ethics Board. The study represents data collected over 6 months (October 2014 to April 2015) through the Kids2Hear program, a student-run initiative at the University of Toronto. As part of the program, medical students were trained by HSC staff in audiological screening and otoscopy. Otoscopy training involved both teaching sessions with OtoSim (an ear training and simulation system) and practice with live subjects .Students from three Toronto elementary schools participated in the Kids2Hear program for the 2014–2015 school year. Consent forms were sent several weeks in advance to the school’s administration to distribute to all students in classrooms whose schedules permitted them to participate. Only those students presenting on the screening day with a consent form completed by their parent/guardian were eligible for hearing screening.Audiological screening consisted of a pure tone screen (30 dB HL) bilaterally assessing frequencies of 1000 Hz, 2000 Hz, and 4000 Hz. Otoscopic inspection was performed bilaterally. The University of Toronto medical students worked side-by-side with professionals from HSC who volunteered to help with the screening sessions. Audiologists supervised medical students conducting audiological screening in a one-to-one ratio, and a staff or resident otolaryngologist confirmed each of the medical student’s otoscopic inspection findings. If a child was identified with deficits or otosopic abnormalities during the screening, Kids2Hear coordinated a referral to 1) the pediatric audiology and otolaryngology clinic at HSC for audiological deficits or 2) a local family physician for children with only otoscopic abnormalities.p < 0.05.Data were analyzed using SPSS version 20 . Descriptive analyses using chi square analyses were used to examine characteristics of the overall sample and assess for gender effect. Significance was assigned at M = 5.8, SD = 1.0) and from kindergarten to grade 3. Table The overall cohort consisted of 228 participants  from three elementary schools in Toronto. Demographic information about the schools and students screened in the Kids2Hear program by school is shown in Table χ2 = 7.78, p < 0.01. Approximately 44 (19.3 %) participants required referral to a family physician for otoscopic abnormalities. Similarly, a greater proportion of females  were identified with otoscopic abnormalities, χ2 = 4.24, p = 0.04. Reasons for referral are shown in Table Table This is an evaluation of a free student-run hearing screening program, “Kids2Hear”, offered to inner-city elementary schools. Our findings show that hearing deficits are common in young children, with about 9.3 % of the overall sample requiring referral for audiology assessment. In addition, 19 % of screened children required referral to a local family physician for otoscopic abnormalities. Occluding cerumen was the most common cause for referral while mucoid and serous effusions were the second most common cause. Furthermore, females were significantly more likely to be identified with either a hearing deficit or otoscopic abnormality. This association has not been previously reported in the literature and warrants further investigation.Risk factors for middle ear effusions are numerous including host factors  and environmental factors  . It is nIn evaluating a screening program, several characteristics must be considered. The major goal of a screening program is to detect a disease at a stage when the treatment can be effective in reducing long-term complications . The disAlthough the overall concept of the Kids2Hear program is in accordance with the values that a screening program should embody, there were several limitations discovered in this study that limited our ability to assess the potential benefits of the screenings performed. The first is that this study includes a relatively small sample size and sample bias, as the schools participating in this program were not picked at random. Secondly, the accuracy of the audiometric methods used in the evaluation of screening for hearing loss should be discussed. The American Speech-Language-Hearing Association guidelines specify that hearing screening must be done with calibrated audiometers, in an environment with sufficiently low ambient noise (<49.5 dB SPL), and minimal distractions in the surrounding environment . The KidLastly, as this was a retrospective analysis, and the elementary students themselves were not enrolled in any form of research study, we were not provided with any baseline health or development information for these children , and we had no ability to follow-up on short or long-term outcomes of the children who were referred for further evaluation. As such, we were unable to correlate the results from the Kids2Hear screening evaluations with the official assessments in the audiology and otolaryngology clinics at HSC, unable to follow up with results and/or management that may have taken place with the local family physicians, and we were unable to track any treatment or interventions that may have taken place . We therefore do not have enough information to assess the potential effectiveness of this screening program at this time.When combining the importance of detecting hearing deficits at an early age (to prevent long-term negative speech and language sequelae) , the facThis is the first evaluation of a student-run free hearing screening program offered at inner-city schools in the literature. Our data demonstrates that hearing deficits and otoscopic abnormalities are common in young children, with 9.3 and 19 % of our overall sample requiring referral for further evaluation. Our findings also indicate that females were significantly more likely to be identified with hearing deficits or otoscopic abnormalities, which has not been demonstrated in the previous literature and could be an avenue for future research. As there was no follow-up on short or long-term outcomes of the children referred for additional evaluation, we currently do not have enough information to assess the potential benefits of this screening program. Additional research is needed to evaluate the Kids2Hear program over additional years to determine the program’s effectiveness.


Development of hydrocephalus can occur after subarachnoid hemorrhage (SAH). Typically, it is diagnosed with computed tomography, CT, scan. However, transcranial sonography (TCS) can be used particularly in patients with craniotomy which removes the acoustic interference of the skull and allows a closer up visualization of brain structures through the skin.We report a 73-year-old woman who was hospitalized for SAH and developed acute hydrocephalus requiring an external ventricular drain (EVD). In this patient, detection and monitoring of hydrocephalus was done and monitored with a small pocket-sized TCS device. Nine days after surgery, weaning of the EVD was attempted. Prior to EVD closure and removal, TCS showed a measurement of the 3rd ventricle at around 1.16 cm. On the third day, the patient deteriorated clinically and the TCS showed a dilated 3rd ventricle measuring 1.37 cm which correlated well with computed tomography and with clinical signs of active hydrocephalus as both her sensorium and communication were affected. Subsequently following EVD re-installation, on the next day, TCS showed that the 3rd ventricle dimension was reduced to 0.99 cm and the following day it went down to 0.69 cm.Patients with SAH and in particular those with a craniotomy can be monitored easily at the bedside with hand-held TCS for the development and monitoring of hydrocephalus. Hydrocephalus in patients with subarachnoid hemorrhage (SAH) is a frequent cause of clinical deterioration. In addition to clinical evaluation, computed tomography (CT) scans are used regularly to monitor for the appearance of hydrocephalus. The use of bedside ultrasound for imaging has expanded to multiple clinical applications especially in the acute critical patient care. We describe a case for which we used a hand-held two-dimensional (2D) transcranial sonography (TCS) probe to follow-up a patient with hydrocephalus after subarachnoid hemorrhage demonstrating high accuracy and clinical correlation. Permission to use the following figures was granted by the patient’s family.A 73-year-old woman was admitted to our hospital in coma after having an acute thunder clap headache. Her vitals were stable apart from high blood pressure and other signs of increased intracranial pressure (ICP). Her CT showed extensive SAH with acute hydrocephalus and signs of elevated ICP. An anterior communicating artery aneurysm was identified on CT angiogram. Patient underwent urgent surgery including insertion of external ventricular drain (EVD), right-sided craniectomy and clipping of the aneurysm at the same time. She had a fair good outcome after the surgery immediately with improvement in consciousness and good control of her ICP. An intensivist with bedside ultrasound experience followed the patient with daily TCS using a hand-held pocket ultrasound device  Fig. a. Image Hydrocephalus is a common problem after subarachnoid hemorrhage SAH . It is ap < 0.01. There were no systematic biais using the Bland–Altman plot. In our case, the craniectomy site allowed direct skin dura matter contact which helps clarify more the brain structures and decreases the distance and eliminates artifacts caused by bone. To our knowledge, so far not many case reports and or case series evoked the possibility to use ultrasound instead of CT scan to monitor these patients’ hydrocephalus. Recently, a case series was published and showed that TCS using B-sonography mode was effective in decision making regarding removal of external ventricular drainage in patients with post-hemorrhagic malabsorption hydrocephalus with good specificity and sensitivity [The accuracy of ultrasound compared to CT scan is not well established in adults and no known large studies confirming or infirming the utility of ultrasound in adults with hydrocephalus. Most of the literature comparing ultrasonography and computed tomography has been done in infants and children because of the natural acoustic windows of the brain through the fontanelles –12. A resitivity , 16.This case illustrates the usefulness of hand-held bedside ultrasound for follow-up of patients with subarachnoid hemorrhage as a non-invasive, simple and safe method to monitor for the development of hydrocephalus and may be to guide management.Patients with SAH and particular those with a craniotomy can be monitored easily at the bedside with hand-held TCS for the development of hydrocephalus. Further studies with larger number of patients should be performed to evaluate the feasibility, sensitivity and specificity of TCS in the detection of hydrocephalus in adult patients after SAH.


Tuber magnatum have not been investigated yet. Volatiles in T. magnatum fruiting bodies were quantitatively and qualitatively determined by both PTR-MS and GC-MS in order to compare the accuracy of the two methods. An electronic nose was also used to characterize truffle samples. The influence of environmental changes on the antioxidant capabilities of fruiting bodies was also determined. Statistically significant differences were found between fruiting bodies with different origins. The relationship between the quality of white truffle fruiting bodies and their specific host plant is described along with an analysis of metabolites other than VOCs that have ecological roles. Our results indicate that the geographical origin  of the fruiting bodies is correlated with the quantity and quality of volatiles and various antioxidant metabolites. This is the first report characterizing antioxidant compounds other than VOCs in white truffles. The correlation between geographical origin and antioxidant contents suggests that these compounds may be useful for certifying the geographical origin of truffles.The influences of various factors, including the symbiosis established with the roots of specific tree species, on the production of volatiles in the fruiting bodies of  The aromatic compounds of fruiting bodies from several Tuber species have been analysed, and more than 200 volatile organic compounds (VOCs) have been studied2. The truffle aroma is derived from a complex mixture of substances from several chemical classes3. The VOCs emitted by truffles may play several roles similar to those reported in plants5 and may be heavily dependent on the environment. This means that VOCs may vary between individual truffles of the same species grown in different locations. Proton Transfer Reaction-Mass Spectrometry (PTR-MS) and multiple factorial analysis of the PTR signals have been used to analyse VOCs and to differentiate between samples of Tuber magnatum Pico fruiting bodies that were collected from two different regions of Italy6. Although the influence of truffles on the physiology of their host trees and on forest ecology has been described, little is known about the effect of the host plant on the VOCs produced by fruiting bodies.Gioacchini, et al.6 and Aprea, et al.7 both used PTR-TOF-MS to analyse white truffle aroma; the consistency of their results suggests that PTR-TOF-MS allows for the direct, accurate and rapid measurement of VOCs even at concentrations as low as a few parts per trillion by volume (pptv). Although PTR-MS is a powerful method, some compounds are ambiguously identified. To more completely describe the volatile compounds of white truffle, our analysis combined the highly sensitive PTR-MS method with gas-chromatography coupled to mass spectrometry (GC-MS) and extended the sampling of fruiting bodies to include various locations in Italy, taking into account the plant species associated with the fungus when possible.Vita, T. magnatum aroma is influenced by various factors including the presence of genetically differentiated populations8 and the variability of the bacterial9 and fungal11 communities associated with the truffle, but the climate of its geographical area of origin has the strongest effect1.The composition of the 12. Antioxidant levels change in response to both biotic and abiotic environmental stresses because, similar to VOCs, they play a role in signalling and in responding to environmental changes. Phenols, ascorbate and glutathione are among the most studied antioxidants that have clear positive impacts on human health13 and delay the oxidative processes that occur in the decay and spoilage of food post-harvest. These metabolites are also involved in defending against biotic and abiotic environmental stresses14. There is considerable information on both the overall antioxidant capability and specific antioxidant molecules in plants and their relevance to plant growth and survival. However, there is currently little information for truffles, although some research is available on the correlation between phenols in truffles and antioxidant properties15. Variations in the contents of other antioxidant metabolites have also been reported in the desert truffle Tirmania nivea collected from different Middle Eastern regions16.Antioxidants are another class of metabolites that vary a lot in response to the environment. They are chemically heterogeneous molecules that interfere with the levels of cellular reactive oxygen species that are generated within the cell as normal by-products of aerobic metabolismWe hypothesized that antioxidant capability and the ascorbate and glutathione contents in truffles may be indicators of geographic origin or plant species association. Therefore, these parameters were analysed to evaluate the relevance of ecological influences on truffle growth.T. magnatum fruiting bodies that were collected from various Italian and Istrian locations and to highlight VOCs that are specific to the different locations. We also attempted to distinguish fruiting bodies taken from the different locations by analysing antioxidant molecules that are influenced by environmental conditions.The purpose of this research is to precisely determine the volatile compounds present in the aroma of T. magnatum was performed with PTR-TOF on selected fruiting bodies , chemical name, and molecular formula. Percentages of VOCs reported in the Supplementary Information  revealed relationships among the PTR-TOF-MS fingerprints of the samples from the nine geographical areas. The coordinates of the nine groups of variables were used to create a map of the groups of compounds Fig.  generallDifferent trends were evident in the contributions of each group of variables to the second MFA dimension. The contributions of terpenes (18.52%) and aldehydes (17.24%) were the most statistically significant. On the other hand, the contributions of esters (3.17%) and other compounds (4.51%) were low. The data from the MFA were also used to determine how samples were separated within the multidimensional space Fig. . They weThe first (F1) and second (F2) factors explained 53.31% and 13.87% of the total variance, respectively. The spatial distributions of the samples in the multidimensional space of the MFA showed that the HRwd (Istria), SMwd (San Miniato), CAsa (Casentino white willow) and CApa (Casentino poplar) samples differed the most. Considering only the first dimension, the samples collected from the Piedmont region  were clearly distinct from most of the samples collected from the Marches and Molise regions. Furthermore, the association of mycorrhiza with oak was a common feature of samples from Alba, Casentino and Sant’Angelo in Vado Fig. .Quantitative data were also visualized using heat maps Fig. , and twoThe GC-MS analyses of volatiles definitively identified 114 compounds, and 1 compound was tentatively identified  to the two first dimensions are summarized in the Supplementary Information Table , as was The quantitative GC-MS data were also visualized with a heat map Fig. , as was The hydrophilic, lipophilic and total antioxidant powers of the truffle samples were analysed. The fruiting bodies collected from different geographical areas had different ratios of lipophilic and hydrophilic antioxidant power. Fruiting bodies collected from Sant’Angelo in Vado (MSqp), Mercatello sul Metauro (MMqc) and San Gimignano (SGwd) had similar levels of lipophilic antioxidant power Fig. , while tAnalyses were also carried out for specific antioxidants including phenols, ascorbate and glutathione Fig. . The ascData obtained by electronic nose give a global information concerning all the volatiles compounds singularly detected by analytical methods. Similarly, the antioxidant capability represents the summation of the antioxidant effects of the single analysed metabolites. On this basis, RDA analysis was performed taking into account these parameters with the aim of detecting the existence of putative correlations between the parameters and between different parameters and truffle samples. Figure Popolus alba) and MSqp and CAqp ; in this latter case, the only conflicting element is represented by the ALqp sample. A deeper statistical investigation of the relationships between single antioxidant parameters and electronic nose data was performed using polynomial regressions to explore correlations between each pair of variables, and calculated based on the whole dataset to highlight those that were most meaningful  and those of northwestern Italy (Asti and Langhe) were genetically distinguishable. Samples from these regions were not included in our study. The climatic parameters for the various geographical areas are shown in the Supplementary Information  and in some cases in terms of the host plant from which they were collected. Rubini, on Table . It shouT. magnatum fruiting bodies. Such a profile had not previously existed for T. magnatum. In addition, some of the volatile compounds that were identified were associated with fruiting bodies from specific regions, and some compounds were unique to a specific origin. Environmental differences between the various sites include many factors, and one factor that possibly affects VOC content is the host-plant, although the impact of geographical origin is more significant.The primary contribution of this work is a comprehensive profile of the VOCs generated by et al.17, these VOCs were defined as “the most important group of odorants” and were detected in the fruiting bodies as well as in the mycelium of T. magnatum and other Tuber species19. These VOCs were also prevalent in our experimental analyses of T. magnatum, which is in agreement with the results from other studies on this truffle21.The white truffle’s aroma is frequently described as sulfurous or garlicky, and this kind of odour is attributed to the presence of sulfur-containing VOCs. After the pioneering work of Fiecchi, Three of the compounds identified methane) were detected in all of the samples by GC-MS. Dimethyl trisulfide was found in the ALqp, MScb, CApa, SMwd, and HRwd samples. Therefore, these sulfides cannot be used to trace the origin of truffle fruiting bodies, but S-allyl-thiopropionate and 3-methylthio-propionaldehyde can. To our knowledge, these two volatile compounds have never been found in the aroma of any truffle species, but our analyses with PTR-TOF identified a higher total number of sulfur compounds.22. Many of these aldehydes are fundamental aroma components23. In our analysis, several of the aldehydes identified were specific to carpophores of definite origins. Aldehyde volatiles were mainly produced by Casentino samples, and they were also found to a lesser extent in Sant’Angelo in Vado fruiting bodies.Linoleic and linolenic acids are important components of biological membranes whose oxidative degradation by lipoxygenase produces aldehydes24 but had never been identified as a T. magnatum volatile. In our experiments, it was emitted exclusively by the Casentino poplar (CApa) sample. The linear aldehyde n-octanal has rarely been reported in T. magnatum26 and was exclusive to the CAsa sample. The longer linear aldehyde n-undecanal (11 carbon atoms) is a common component of essential oils of Citrus spp. and fruit juice27, but it has never been found in truffles. We found that it was produced by the CAsa samples but not in any of the others.(4Z)-decenal was reported for the first time in orange essential oil28, but never in Tuber spp. Our analysis of the samples from MSqb and HRwd revealed that (2E)-butenal is produced in T. magnatum , and this was confirmed by PTR-TOF results.(2E)-butenal has been detected in kiwi fruit essence and in other fruitset al.22, these aldehydes do not originate from lipid oxidation but from Strecker degradation of amino acids. The presence of the very similar compound (same molecular weight), 2-methylpent-2-enal, was detected in Tuber borchii29 and T. magnatum26. Other aldehydes, namely, 2-methyl pentanal, ligustral and benzaldehyde, occurred only in MMcb and SMwd samples. In a previous paper6, we reported that some volatile compounds are able to discriminate Alba from San Miniato samples. Some of these compounds  were also found in this analysis, but only in San Miniato fruiting bodies . 1-octen-3-ol is believed to act as signal to plants19. In our study, this compound was detected in fruiting bodies of various origins, and its occurrence was also not dependent on the host plant; hence, it is not useful for identifying a truffle’s origin.In previous work, specific attention was given to the alcohol 1-octen-3-ol. For example, Combet, e.g., the C12 linear alcohol dodecanol in Casentino oak, CAqp). On the other hand, Casentino poplar (CApa) emitted both branched and linear volatiles as ethyl hexanol and (4Z)-decen-1-ol. Pennazza, et al.26 have reported the occasional occurrence of (4Z)-decen-1-ol in their stored white truffle fruiting bodies, which are naturally harvested in Marche, close to the Casentino area. In our analyses, this compound occurred only in CAsa and SMwd samples. SMwd, on the other hand, was the only sample to produce nonylol, a C-9 alcohol never found before in truffles. In our study, the volatile propyl alcohol was detected in the ALqp and ALpa samples, which has also never been reported in T. magnatum. Another alcohol, n-heptanol, was found only in ALpa by GC-MS but was revealed by PTR-TOF analysis to be in this sample as well as many others . The acquisition of data with two different instruments that have different specificities and sensitivities provided a greater degree of confidence in the final results.Other alcohols were found to be linked to specific mycorrhiza  mycorrhiza than in pine mycorrhiza.et al.2 reported that 3-octanone and 1-octen-3-ol are unique to the fruiting bodies of T. uncinatum of Italian origin and are not found in those from Switzerland and France.In addition to the influence of the host plant, the truffle’s origin also determines which volatile compounds it produces. Splivallo, T. magnatum26 but only at very low levels. We found a similar compound, 6-methyl-2-heptanone, was emitted by T. melanosporum. The ketone 2-decanone, a compound with an orange-like floral odor that has been detected in several plants36, has never been reported in T. magnatum. Another ketone, 2-undecanone, was almost specifically produced by CApa. This volatile has been detected in the aroma of several truffles, including T. melanosporum and T. aestivum33 and T. indicum31, but has never been found in T. magnatum. SMwd samples also had a specific ketone, 2-tridecanone, which has so far only been reported in field-grown onion37.We found the volatile compound 6-methyl-5-hepten-2-one in CApa and SMwd. This volatile compound has also been detected in Since PTR-TOF analysis detected only one terpene, our discussion is limited to the GC-MS data. For the PTR-TOF-MS data, compounds that have the same molecular formula cannot be reliably identified.38. They have been investigated less in truffles, although their occurrences in the aromas of fruiting bodies have been reported29. However, they are often limited to very few compounds such as the cyclic monoterpene (molecular formula C10H16) limonene or the related alkyl benzene p-cymene.Volatile terpenes are major constituents of the plant volatilome and more importantly plant essential oils31, no molecules belonging to terpenes were reported. However, research on T. magnatum by Gioacchini, et al.1 indicated that white truffle aroma had a particular composition, which appeared to consist of only two main groups of volatiles: sulfur compounds and terpenes. In their study, which included fruiting bodies harvested in six Italian regions, they listed 28 sulfur compounds and 24 terpenes. Such discrepancies between reported data clearly show that a comprehensive description of the volatiles of white truffle is still far from complete.In the most complete inventory of volatile compounds for fruiting bodies of three truffle speciese.g., limonene, and α- and β-pinene). However, other monoterpenes are uniquely produced by fruiting bodies of a specific origin or host-plant. Some monoterpenes, such as α-terpinene, eucalyptol and camphor, have been detected in Casentino fruiting bodies with mycorrhiza either on poplar (CApa) or on willow (CAsa). A wide variety of terpenes was found in the samples from Casentino, which agrees with the results of Gioacchini, et al.1. It is worth noting the proximity and the substantial similarity of the tree species that make up the forests of the Casentino and Marche areas where those authors collected their samples.Our results indicate that some terpenes are almost ubiquitous in the white truffle volatilome  samples are from the Marche region. Additionally, the climatic data for the Casentino and Marche geographical areas are similar .The occurrence of this class of volatiles in the fruiting bodies of several truffle species, including white truffle, is well knownet al.31, esters occur in the aroma of several species of truffles, but no esters have been reported in T. magnatum aroma. We found 14 esters primarily in the samples from Tuscany and Marche. Some of them, such as vinyl acetate, were uniquely produced by CAqp, which was confirmed by PTR-TOF.As reported by Splivallo, 41; 3,5,5-trimethylhexyl-acetate is a volatile with a floral-sweet odour, and isobutyl pentanoate contributes to the odour of several apples42. Other volatile esters were detected specifically in the fruiting bodies from MMqc  and from MSqb and MScb. 3-acetoxyoctane was identified for the first time in MSqb truffles. In MScb, isobutyl-isobutyrate, which is characterized by a pleasant fruity odour, was detected, and it is present in nearly all fruits.Several esters were identified in the CApa fruiting bodies’ aroma: (2E)-hexenyl acetate is a common component of the volatilome of several fruits and mushrooms; ethyl lactate is a chemical that conveys a strawberry taste43.The SMwd sample was particularly rich in esters; nine compounds were identified. This great variety of esters in San Miniato truffles is crucial for their characterization, and it may also play an important role in defining their aroma. Only low molecular weight esters, such as those found in the SMwd samples, have a pleasant odour. In particular, the low molecular-weight esters of butyric acid (similar to esters found in SMwd) have a pleasant aroma and taste44.Antioxidant power is a measure of a compounds ability to detoxify reactive oxygen species produced within cells. Several molecules serve as antioxidants, of which phenols and polyphenols play a key role. It is worth noting that the methods used to extract metabolites from fruiting bodies do not result in the disruption of bacterial and yeast cells, which are the most abundant microorganisms colonizing fruiting bodies. Therefore, the profiles of antioxidant molecules that we have described are truly derived from truffle fruiting bodies. Unlike for plants, where many papers describe the antioxidant properties of different tissues and organs, there are very few studies on antioxidants in truffles or on the contribution of phenols to antioxidant activity in mushrooms16, who found a significant correlation between phenols and antioxidant capacity in the desert truffle Tirmania nivea collected from four different Middle Eastern regions16. In the truffles analysed here, neither the levels of glutathione nor those of ascorbate reflect the antioxidant power of the truffle fruiting bodies. This indicates that the total antioxidant activity is the result of various molecules present in different amounts that have different specificities for scavenging the various reactive species produced within cells.Our results suggest that phenols are not the main component conferring antioxidant properties to truffles, since no correlation was found between phenol content and antioxidant power in the fruiting bodies. The samples with the highest antioxidant powers were not the same as those with the highest phenol contents. This is in contrast with the results of Al-LaithTuber huidongense45. The amounts of ascorbate found in the fruiting bodies are much lower than those found in most of the metabolically active plant tissues, although there is considerable variability in antioxidant content between plant species, tissues and growth conditions14. Very small amounts of ascorbate have been found in desert truffles. In the fruiting bodies of T. nivea, the levels of ascorbate range between 5.9 and 11.4 mg/100 g of dry weight (which corresponds to 0.3 and 0.6 nmoles/g of dry weight)16. Similarly in Terfezia boudieri, the ascorbate content is 12.2 mg/100 g of dry weight (approximately 0.6 nmoles/g of dry weight)46. These values are more than 100–200 times lower than those found in T. magnatum (between 50 to 120 nmol/g of fresh weight). It should be emphasized that in these desert truffles, ascorbate was quantified using a colorimetric method based on the redox capability of ascorbate, while here, an enzymatic and indeed more specific method has been used. The presence of ascorbate metabolism in fungi is still under debate. Ascorbate has been reported in a few genera of yeast, but not in others48. According to a recent study on the evolution of the ascorbate biosynthetic pathways in eukaryotes, genes with homology to the sequences of the last enzymes in the eukaryotic (both photosynthetic and not-photosynthetic) ascorbate biosynthetic pathway are also present in some unicellular and multicellular fungi49. It is also known that heme-peroxidases  are widely distributed in all domains of life including fungi50. However, ascorbate was not detected in the fruiting bodies of the edible Basydiomicota Pleurotus eryngii when it was analysed with the same methods used here . More in-depth studies are necessary in order to determine which phyletic lines of the Fungi kingdom are capable of ascorbate biosynthesis and how this ability was acquired by truffles. On the other hand, glutathione is a known part of truffle redox metabolism. Several glutathione-related genes have been identified in fungi including in T. melanosporum (the sequence of which is available at http://mycor.nancy.inra.fr/IMGC/TuberGenome/). It has also been suggested that glutathione appeared during evolution when oxygen increased in the atmosphere. It is likely that this was the first metabolic strategy for coping with oxygen-dependent oxidation51. Developmental phases could also strongly affect the levels of antioxidant metabolism as they do in other organisms, since both ascorbate and glutathione participate in the cell cycle and in other metabolic processes relevant to cell differentiation and organism growth14. This could be the focus of future studies.A recent study reported that some polysaccharides strongly contribute to the antioxidant/redox activity of Truffles collected in the same geographical area such as those from Alba and Aquila have some similarities in their lipophilic and hydrophilic antioxidant properties. However, the ecological environment where the truffles grow also has a strong influence on their antioxidant capability, since statistically significant differences were observed for antioxidant properties between samples that were collected in the same geographical area but had established mycorrhizal symbioses with different plants. This is even more evident in the phenol, ascorbate and glutathione contents, since their values varied significantly among the samples collected in the same area but near different plants. The results are in line with the physiological role of these metabolites in the organism / environment interaction. The climatic data are partially correlated with antioxidant contents. In fact, the highest level of hydrophilic antioxidant power and total glutathione was observed in the Casentino truffles associated with willow (CAsa), while total phenols were high in all Casentino truffles Fig. .et al.52, that have compared two different sets of data, we have used antioxidant measurements and electronic nose data together to differentiate samples.The RDA results demonstrate how we could differentiate samples using two classes of independent variables. Similarly to Falasconi, 53.The polynomial regression models show how the lipophilic antioxidant power is well correlated with the data of three nose sensors. It is worth noting that a consistent part of the identified VOC, such as terpens, are mostly lipophilic, therefore this correlation might suggest that part of the lipophilic antioxidant power is conferred by VOCs with lipophilic properties. Consistently, volatiles isoprenoids and phenylpropanes have been suggested to confer antioxidant protection under prolonged stressNevertheless, the use of a large number of samples could help to develop a system for identifying the origin of unknown truffles using this alternative, fast and reliable method which includes different kinds of analyses. In summary:a) We used three methods  to analyse the VOCs from white truffles, and to the best of our knowledge, we produced more accurate results than have previously been possible. Our findings also support the idea that volatiles, both individually and as a group of similar molecules, could be used to characterize the specific origin of truffle fruiting bodies. For example, a class of esters was unique to truffles from San Miniato, one of the Italian areas with higher quality white truffles, while acetyl isovaleryl and terpinolene were found in Istrian truffles  The host plant has some influence on the types of volatile compounds emitted from truffles with the same origin. Alcohols and aldehydes such as decenol and decenal or n-octanal and n-undecanal are produced only by CApa and CAsa, respectively see Figs  and 2B.T. magnatum indicates that sulfur compounds contribute the most to the formation of the truffle’s aroma methane and dimethyl sulfide). Several VOCs belonging to the terpene class  are present specifically in the white truffles of central Italy.c) Our analysis of individual VOCs in d) The antioxidant properties of fruiting bodies that were collected from geographically distant areas were different, and this may be attributed to the specific mycorrhizal symbiosis Fig. .e) The RDA model appears useful to discriminate samples based on their geographical origin/ host plant. Further analysis confirmed the existence of a precise correlation between lipophilic antioxidant power and electronic nose results.As a general comment, we consider it appropriate to mention that inside of the fruiting bodies, other fungi such as yeasts and bacteria can contribute to the biochemical features of truffles. Therefore, it is possible that some of the VOCs identified are not actually produced by the truffles.T. magnatum Pico fruiting bodies were collected from various areas of Italy and Croatia  are included in the Supplementary Information , two from Marches , one from Piedmont (Alba), one from Molise (Isernia), one from Abruzzo (Aquila), and one from Croatia . The symbiotic plant species were known for several of the samples, including Casentino ; Mercatello sul Metauro ; Sant’Angelo in Vado ; and Alba . Together, the total number of samples analysed was 85 (17 × 5) per year. The samples were analysed separately. Detailed sample information is shown in Table Samples of on Table . SpeciesThree fruiting bodies were used for the MS analysis for each source and year (n = 6), and 2 fruiting bodies were used for the analysis of the antioxidant compounds for each origin and year (n = 4).All samples were stored at 4 °C after collection, and all of the analyses were completed within three days. When it was not possible to comply with this requirement, even for a single year of experimentation, the samples were excluded from the analysis to avoid any alterations due to sample deterioration.When possible, samples from the same geographical site were analysed on the same day.The full details for PTR-TOF, GC-MS/ GC-FID analysis, antioxidant activity, antioxidant compound analysis, PCA and electronic nose measurements are in the Supplementary Information Materials and Methods.Information related to the statistical analyses described in this article are reported in the Supplementary Information Materials and Methods.Supplementary Information


The objective of this study is to investigate whether chronic inflammatory demyelinating polyneuropathy (CIDP) and its subtypes differ in their type 1 T-helper (TH1) cell response against nodal/paranodal neurofascin  as well as myelin protein zero (P0 180–199) and myelin basic protein (MBP 82–100).n = 18), distal acquired demyelinating polyneuropathy (n = 8), multifocal acquired demyelinating sensory and motor polyneuropathy , and sensory CIDP (n = 13) compared to other non-immune polyneuropathy  and healthy controls (n = 9).Interferon-gamma (IFN-γ) enzyme-linked immunospot assay was used to detect antigen-specific T cell responses in 48 patients suffering typical CIDP  and were found in 77% of CIDP patients. Patients with limited antigen-specific response were females, more severely affected with neuropathic pain and proximal paresis. The area under the receiver operating characteristics curve (AUC) of NF186 in MADSAM was 0.94 [95% confidential interval (CI) 0.82–1.00] compared to ON. For sensory CIDP, AUC of P0 180–199 was 0.94 (95% CI 0.86–1.00) and for MBP 82–100 0.95 (95% CI 0.88–1.00) compared to ON.Cell-mediated immune responses to (para)nodal and myelin-derived antigens are common in CIDP. TH1 response against NF186 may be used as a biomarker for MADSAM and TH1 responses against P0 180–199 and MBP 82–100 as biomarkers for sensory CIDP. Larger multicenter studies study are warranted in order to establish these immunological markers as a diagnostic tools. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune disorder of the peripheral nervous system and can be divided clinically into typical CIDP and atypical variants, such as distal acquired demyelinating polyneuropathy (DADS), multifocal acquired demyelinating sensory and motor polyneuropathy (MADSAM), and sensory CIDP , 2. Due There is an emerging body of evidence that molecules of the nodal/paranodal complex may be essential targets for blocking propagation of nerve impulses along myelin fibers . Thus, aThe study was approved by the ethical committee of Charité University Medicine Berlin. All patients were recruited in the outpatient clinic of the Charité Department of Neurology. All patients gave their written informed consent for the study. Pseudonyms were used for the study.n = 18), DADS (n = 8), MADSAM (n = 9), and sensory CIDP (n = 13) were recruited. Diagnoses were made according to the criteria of the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) (n = 13), metabolic or toxic polyneuropathy (n = 1), hereditary polyneuropathy (n = 1) or diabetic polyneuropathy (n = 2), motoneuron disease (n = 1), ATTR amyloidosis-associated neuropathy (n = 1), and nine healthy controls (HCs). Clinical and experimental data of one patient have been submitted as a case report elsewhere  , and theFNS/PNS) . For claFNS/PNS) , summariFNS/PNS) , 17. As g for 20 min and diluted in CTL-Test-Medium  at a concentration of 6 × 106 cells/mL.For ELISPOT, blood samples were collected using CPT tubes . In patients treated with intravenous immunoglobulins (IVIg), blood was obtained on the first day of IVIg-therapy before starting the infusion. Peripheral blood monocytes (PBMCs) were isolated within 3 h by density gradient centrifugation at 1,500 5 cells/well in presence of anti-CD28 antibody which enhances the costimulatory signal  at a concentration of 2 μg/mL and conjugated at 1:1,000 to streptavidin-horseradish-peroxidase . Plates were developed with 3-amino-9-ethyl carbazole reagent . The resulting spots were detected, counted and analyzed via Elispot Reader  and appendant iSpot 04 Software. Spot forming units (SFU) for each antigen triplicate were averaged and subtracted by average SFU of spontaneous IFN-γ secretion and then calculated for a cell quantity of 106 cells. Analyzing of the data was performed in a blinded fashion.For ELISPOT assay, we used our established protocol  based ony signal   and were described earlier . P0 180–U-test, when suitable. Fisher’s exact test was used to compare frequencies of the non-metric clinical parameters sex, response to IVIg-treatment, CDAS, tremor, sensory ataxia, neuropathic pain, asymmetric paresis, proximal paresis, drop foot, distal motoric latency, F-wave latencies, nerve conduction velocities, conduction block, and positive CSF. We calculated Spearman’s rho to analyze the relation of P0 to MBP. In order to evaluate the discrimination properties of the different antigen T cell responses for differentiating a specific type of CIPD from ON or other atypical CIPD variants, we calculated the area under the curve (AUC) and 95% confidential interval (CI) from receiver operating characteristics (ROC)-curve analyses. Good discriminative potential of an antigen response in a CIDP subtype compared to the control group was defined as an AUC > 7, which did not include 0.5 in 95% CI. To achieve higher discrimination properties, we also analyzed combinations of promising markers using multiple logistic regression with regard to the discrimination of specific disease types from ON. Statistical analyses were performed with GraphPad Prism version 6.0 . No adjustment for multiple testing was made. A p-value <0.05 was considered significant.We compared clinical baseline measurements  as well as antigen-specific IFN-γ responses using Kruskal–Wallis test followed by Dunn’s multiple comparison test or Mann–Whitney post hoc defined cutoff values .post hoc defined cutoffs . Clinical disease activity status, however, improvement upon IVIg-treatment, or clinical features such as tremor and ataxia were independent of T cell reaction specific to NF155, NF186, P0 180–199, or MBP 82–100 (Table Asymmetric paresis was seen in the NF186 responsive group. The MBP responsive group was male and showed a shorter disease duration compared to the MBP-unresponsive group. Similarly, patients responsive to P0 were male and younger than the P0 negative group. Looking at demyelinating parameters, we found by trend an association between the NF186 response and conduction blocks (In the present study, we demonstrated specific IFN-γ T cell responses against the paranodal/nodal antigens NF155 and NF186 as well as against myelin-derived antigens MBP 82–100 and P0 180–199 in CIDP compared to ON. Positive IFN-γ responses against two or more antigens were highly predictive for any subtype of CIDP, with MADSAM and sensory CIDP showing the broadest immune response to the four tested antigens. ROC analysis indicated highest discriminative potential of NF186-specific T cell responses in MADSAM and the highest discriminative potential of P0 180–199 and MBP 82–100-specific response in sensory CIDP, suggesting the presence of cell-mediated immune responses against these antigens as a suitable biomarker for CIDP diagnosis.The autoreactive T cell responses against NF155 and NF186 that we demonstrated here for typical CIDP and MADSAM might be of particular relevance. Recently, IgG4 antibodies to NF155 have been found in a clinically distinct subgroup of CIDP that show a younger age at onset, tremor, ataxia, CNS demyelination and a poor response to IVIg treatment which was not compatible with established EFNS classification criteria , 23. BasIn the present study, we detected IFN-γ responses against P0 and MBP in 67% of our CIDP cohort. P0 is one of the major peripheral myelin proteins that functions as autoantigen in models of autoimmune peripheral neuropathy , 25. TheIn our cohort, eleven CIDP patients showed only a limited antigen-specific response. Looking at their clinical characteristics, we found these patients were older, had a longer disease duration, a lower MRC, and increased INCAT score with manifest long-term damages such as severe paresis and pain syndromes. Thus, the lack in autoimmune responses in this non-responder group could be attributable to a somewhat “burnt-out” disease status. We observed asymmetric paresis in the NF186 responsive group, which is compatible with the clinical definition of MADSAM patients. Patients responding to MBP and P0 were male and younger than those in the non-responsive group. This concurs with a recent clinical description of sensory CIDP . In contIn summary, we provide evidence for underlying auto-reactive T cell immune responses against neurofascin as well as compact myelin epitopes in CIDP and variants. P0 180–199 and MBP 82–100 IFN-γ responses were associated with sensory CIDP, whereas NF186-specific IFN-γ response was associated with MADSAM. Further multicentric studies including other inflammatory neuropathies are required to validate these promising biomarkers with reliable cut offs as useful diagnostic tools for CIDP, its subtypes, and for treatment guidance.The study was approved by the ethical committee of Charité University Medicine Berlin. All patients were recruited in the outpatient clinic of the Charité Department of Neurology. All patients gave their written informed consent for the study. Pseudonyms were used for the study.J-MD: acquisition, analysis, and interpretation of data, writing of manuscript. MS: acquisition and analyses of data. CM: analyses of data and critical revision of manuscript for intellectual content. KH, patient recruitment, critical revision of manuscript for intellectual content. EM, provision of peptides, analysis, and interpretation of data. AM: critical revision of manuscript for intellectual content, conduct of study, and supervision. JK: study concept and design, patient recruitment, analysis and interpretation of data, study supervision, and writing of manuscript.J-MD, MS, and CM report no disclosure. KH has received speaker honoraria or refunding of conference travel costs from Abbvie, Astellas, CSL Behring, Gilead Sciences GmbH, Pfizer, ViiV Healthcare GmbH. AM reports grants from Grifols during the conduct of the study; personal fees from Grifols and Octapharma. EM received grant support from Novartis and personal compensations from Novartis, Genzyme, and Roche. JK was supported by a Rahel-Hirsch scholarship from the Charité University Medicine, Berlin. She received personal compensation for speaker fees and advisory boards outside the submitted work from Grifols, Octapharma and CSL Behring.


Objective, To survey the pediatric trauma programs to ascertain if and how etomidate is being used for rapid sequence intubation (RSI) in pediatric trauma patients. Design, A 25 question survey was created using REDCaps. A link to the survey was emailed to each of the pediatric and adult trauma programs that care for pediatric patients. Setting, Pediatric trauma programs and adult trauma programs caring for pediatric patients. Intervention, None. Measurements and Main Results, A total of 16% of programs responded (40/247). The majority of the centers that responded are urban, academic, teaching Level 1 pediatric trauma centers that provide care for > 200 pediatric trauma patients annually.  The trauma program directors were the most likely to respond to the survey (18/40).  33/38 respondents state they use etomidate in their RSI protocol but it is not used in all pediatric trauma patients.  26/38 respondents believe that etomidate is associated with adrenal suppression and 24/37 believe it exacerbates adrenal suppression in pediatric trauma patients yet 28 of 37 respondents do not believe it is clinically relevant. Conclusions, Based on the results of the survey, the use of etomidate in pediatric trauma patients is common among urban, academic, teaching, level 1 pediatric trauma centers.  A prospective evaluation of etomidate use for RSI in pediatric trauma patients to evaluate is potential effects on adrenal suppression and hemodynamics is warranted. Pediatric trauma patients are often intubated using rapid sequence intubation (RSI) with an anesthetic and paralytic agent. Induction agents include narcotics, benzodiazepines, non-benzodiazepine hypnotics, and barbiturates. Etomidate is an imidazole-derived ultra-short acting non-barbiturate hypnotic . PotentiContinuous intravenous administration of etomidate has been associated with adrenocortical dysfunction (ACD) and increased patient mortality in adults ,4,5,6. SA 25 question survey  was creaCorrespondence regarding the survey was sent to a total of 247 programs that provide care for pediatric trauma patients in the United States. A total of 40 responses were received for a response rate of 16.2%. The majority of the centers, , that responded were Level 1 academic, teaching, pediatric trauma centers that describe themselves as located in an urban  setting that provide care for >200  pediatric trauma patients annually . The traFifteen  respondents indicated that between 25 and 49 pediatric trauma patients are intubated annually followed by 13  respondents indicated they intubate >50 pediatric trauma patients annually. Twenty-three  respondents state they provide care for pediatric trauma patients in a separate trauma bay within the emergency department (ED) and the airway is primarily managed by emergency department (ED) personnel  followed by anesthesia  . Thirty-The survey response rate of 16.2% was low but not unexpected when surveying medical and program directors . Within p < 0.05). In a retrospective study, den Brinker found a similar decrease of cortisol levels in children with meningococcal sepsis given a single dose of etomidate for RSI [Despite the potential concern(s), etomidate use in RSI appears to be more frequent than originally expected. One of the most concerning adverse effects with etomidate is a reduction in cortisol production resulting from inhibition of 11-β-hydroxylase within the adrenal cortex . ACD was for RSI . Sixty cet al. published a review of 77 children that received etomidate for RSI in a pediatric ED [In 2000, Sokolove reviewed 100 children <10 years of age who received etomidate for RSI . No chilatric ED . The meaatric ED .The range of conditions and diagnoses associated with pediatric trauma patients varies widely. Despite the concern for ACD in the aforementioned studies, etomidate use seems to be common among pediatric trauma programs in the United States. While clinicians can diagnose sepsis and shock upon presentation, it is rather difficult to know which, if any, trauma patients will progress to meeting sepsis and/or shock criteria after initial presentation. Interestingly, approximately 65% of respondents do not believe ACD is clinically relevant, specifically in pediatric trauma patients. However, none of the respondents check cortisol levels in any of their pediatric trauma patients to assess whether ACD is present or significant.Typically, there are several questions that clinicians feel need to be answered regarding etomidate. The first is whether etomidate inhibits cortisol production. Pharmacologically, inhibition of 11-beta-hydroxylase occurs after etomidate administration. Secondly, does this inhibition lead to detectable decreases in cortisol in pediatric patients? There appears to be detectable decreases after etomidate administration but in pediatrics. However, observational study methodology makes this difficult to discern . Lastly,Limitations of our survey and the generalizability of its findings include a lower response rate, which introduces the potential for non-response bias. A small sample of respondents could limit the interpretation of any significant relationship for the use of etomidate for RSI in certain clinical scenarios. In order to develop a survey that practitioners would be able to complete in a timely manner, the survey was only able to query basic information regarding the use of etomidate for RSI for pediatric trauma patients. Interest in the use and potential consequences of etomidate in the pediatric trauma population could be heightened through the creation of a working group in a professional society and/or a practice based research network (PBRN). More detailed information, perhaps a medication use evaluation (MUE), could be conducted with a larger, more in-depth survey. Respondent bias could also have affected the response rate in this survey because clinicians who do not use etomidate may not be as likely to complete the survey as are clinicians who do use etomidate. There may also be differences between self-reported practice and actual practice. Further, as an exploratory pilot study, the purpose of the survey was to evaluate the feasibility of conducting future research on etomidate use in pediatric trauma patients. Assessing the practical aspects of carrying out a larger scale survey and/or prospective clinical research study is paramount.In this self-selected sample, the use of etomidate in pediatric trauma patients undergoing RSI is common among urban, academic, teaching, level 1 pediatric trauma centers. A retrospective MUE or case series coupled with a prospective evaluation of etomidate use for RSI in pediatric trauma patients to evaluate its potential ACD, hemodynamics, and outcomes is warranted.


We explored adherence to the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) guidelines for the diagnosis and treatment of chronic inflammatory demyelinating polyneuropathy (CIDP) by reviewing data from a specialty pharmacy database.Clinical and electrophysiologic data were reviewed for 65 consecutive patients treated with intravenous immunoglobulin (IVIG) for CIDP. Three neuromuscular neurologists independently classified cases according to EFNS/PNS criteria as (1) fulfilling CIDP criteria; (2) non‐CIDP ; or (3) unknown (insufficient information).Patients were treated by 31 different community neurologists in 14 states. Only seven patients (11%) met clinical and electrodiagnostic CIDP criteria. The remainder (89%) did not have CIDP (49%) or were unknown (40%). IVIG mean induction dose was 1.25 g/kg, mean maintenance dose 0.79 g/kg, and mean interval between infusions was 23 days.Adherence to EFNS/PNS CIDP diagnostic and treatment guidelines in the general neurologic community was poor. Improved education and awareness of widely available CIDP guidelines are recommended. All patients signed a waiver of consent.2.2® database .Pertinent positive and negative data, including IVIG dosing information, were compiled and anonymized by one reviewer (JAA) and recorded without interpretation into a CareLogicThree reviewers used EFNS/PNS guidelines to independently evaluate each case. “Typical” and “atypical” variants were included. Typical CIDP was defined as chronically progressive, stepwise, or recurrent symmetric proximal and distal weakness and sensory dysfunction of all extremities, developing over at least 2 months, with possible cranial nerve involvement and diminished or absent deep tendon reflexes in all extremities . However, 19 neurologists referred one patient, and one neurologist contributed 19 patients.3.2Interpretation of clinical and electrophysiologic data by reviewer is shown in Table 3.3Only 11% (7/65) of cases had sufficient documentation to confirm a diagnosis of CIDP  often were not recorded, electrophysiologic studies were inadequately performed or interpreted, and supportive diagnostic studies were not carried out, yet these patients were diagnosed and treated as CIDP. We also observed that based on the available data there was high diagnostic certainty that at least 49% did not have CIDP. These observations are disconcerting. We appreciate that treatment trials may have been reasonable in some with equivocal diagnostic findings or when a clear distinction between other immune‐mediated neuropathies cannot be made, as in the case of the patient reclassified as MMN. However, the majority of misdiagnosed patients had no such uncertainty. Approximately half of the patients had clinical, electrophysiologic, or exclusionary data that unequivocally should have precluded the diagnosis of CIDP.We acknowledge that inclusion of 19 patients from one provider, none of whom had CIDP, influenced the percentage of patients in each diagnostic category. When these patients were excluded, the frequency of confirmed CIDP increased to only 15%, and 37% did not have CIDP. We elected to include the 19 misdiagnosed patients from the single provider into our analysis for several reasons. First, our intention was to report a real‐world sample of consecutive patients from a specialty pharmacy for home infusion of IVIG. Second, it raises concerns that while the overall frequency of CIDP misdiagnosis is high, there may be regional pockets and individual practitioners within the United States that are more prone to overdiagnosis. This outlier may call attention to a systemic issue that is not yet fully appreciated.The frequency of CIDP misdiagnosis previously has been reported to be as high as 47%  are thus included as “CIDP definite, probable, or possible,” then the percentage of confirmed cases increases from 11% (7/65) to 17% (11/65). We consider this an unlikely explanation for our findings because in all referring clinical notes the codes matched the documented clinical conclusion.All patients in this series were treated for presumptive CIDP, yet 70% of IVIG loading doses and 61% of maintenance regimens were below evidence‐based trial data , a “possible” diagnosis improves sensitivity to 91%, albeit at the cost of specificity (66%) (Breiner & Brannagan, We appreciate that because our data were collected from a single specialty pharmacy, it may not be representative of the overall level of care of CIDP in all parts of the United States or by all clinicians that take care of patients with CIDP. With this caveat in mind, our findings do provide useful insight into the diagnostic and treatment practices in one real‐world setting. The extent to which these findings can be systemically extrapolated requires further study.5Chronic inflammatory demyelinating polyneuropathy is commonly misdiagnosed and insufficiently treated with IVIG in general community practice, at least according to the documentation used to obtain approval for IVIG treatment. Widely accepted and readily available CIDP diagnostic and treatment guidelines are underutilized during routine clinical care, highlighting the need for improved education and awareness of existing guidelines. Review of the records showed poor documentation and interpretation of bedside neuromuscular and electrodiagnostic findings. This study raises concerns about the adequacy of neurological training required for appropriate management of neuromuscular diseases. We urge greater adherence to published diagnostic criteria and proper use of proven treatment protocols when caring for patients with CIDP.None declared.