The known neurotropism of the Zika virus (ZikV) suggests that auditory organs and their neural pathways may be affected by prenatal Zika infections. Among the possible manifestations are audiological and language disorders, but so far, the data in the literature are inconclusive. Objective: To describe early and late hearing disorders in children with Congenital Zika Virus Infection (CZVI) and evaluate the language development of this population between 14 and 47 months of age and its possible correlation with the alterations found in auditory exams. Methods: Longitudinal, prospective, observational study of newborns born in Juiz de Fora and its macroregion with confirmed diagnosis of ZikV infection during pregnancy. Participants were examined from one to four years of age for hearing using the transient otoacoustic emissions (TOAE) test, immittance testing and brainstem auditory evoked potential (BAEP), and language using the Bayley Scales of Infant Development—Third Edition (Bayley III). Results: 15 participants were included; eight (53.33%) presented alterations in at least one of the hearing tests, one had an early loss (6%) of sensorineural origin, and seven (46.67%) had a poor language performance. In the three (20%) participants whose audiological exams were altered, there was language impairment, and two (13.33%) participants had extensive malformations in the central nervous system (CNS), presented language delay, and hearing exams were within normality. Conclusion: Infants and preschoolers with CZVI may present early neurosensory loss and late hearing loss with fluctuating character. Even if there were no significant association between the audiological exams results and the Bayley III performance, in the present sample, language development was below expectations for the age in the participants who had alterations in the three audiological exams, when there is early hearing loss or extensive lesions to the CNS. The results reinforce the importance of audiological examinations, especially the BAEP morphological and auditory threshold, in monitoring cases of CZVI until at least three years of age. A Public Health Emergency of International Concern was declared by the World Health Organization in February 2016 . This evThe known neurotropism of the ZikV  suggestsIn a retrospective study, a complete assessment of hearing function in a series of 70 children with microcephaly and laboratory evidence of CZVI showed that four (6%) infants had sensorineural hearing loss. The examination of otoacoustic missions (OAS) is performed as a neonatal screening test and the frequency-specific brainstem auditory evoked potential (BAEP) is performed one month later to confirm the loss . HoweverThe review on hearing findings associated with ZikV infection by Barbosa et al.  concludeConsidering the lack of prospective longitudinal studies that thoroughly investigate hearing and language in a population of preschoolers proven to be exposed to Gestational Zika Virus Infection, the objective of the present study is to describe early and late hearing disorders in children with CZVI and evaluate the language development in this population between 14 and 47 months of age and its possible correlation with the alterations found in auditory exams.A longitudinal, prospective, observational study was carried out. This is part of a larger project entitled: “Gestational infection by Zika Virus: child development from zero to seven years of age, environmental context and epidemiological profile”, which was approved by the Ethics Committee in Research on Human Beings of the Federal University of Juiz de Fora (UFJF), Juiz de Fora, Minas Gerais, Brazil, under opinion np. 2,001,169.Newborns born from November 2015 to March 2018 in Juiz de Fora and its macroregion, who had a confirmed diagnosis of ZikV infection during pregnancy, were included. Cases that presented genetic syndromes, progressive diseases or congenital toxoplasmosis, rubella, cytomegalovirus, HIV, herpes, and syphilis were not included.Of the 19 potential participants in Juiz de Fora and its microregion , 15 presThe diagnosis of GZVI was made during pregnancy on the participants’ mothers using RT-PCR for cases where blood collection was performed until the fourth day of the clinic period  ,22, or tParticipants were assessed longitudinally for hearing and language at different times between 14 and 47 months using the instruments and tests described below. To assess hearing, three instruments were used: the transient otoacoustic emissions test (TOAE), immittance testing, and the brainstem auditory evoked potential (BAEP). The TOAE recording was performed by the Otodynamics Otoport Lite equipment, which uses the non-linear click stimulus predominantly in the frequency range between 1000 to 4000 Hz, with an intensity of 64 dB pe NPS. The emission analysis criterion was a 6 dB SNR signal/noise ratio in three out of five tested frequency bands. A total of 260 sweeps of 16 stimuli were used. The maximum test time was 300 seconds, and the recording frequency bands of the device were 1.0 1.5, 2.0, 3.0, and 4.0 kHz. The parameters used to consider the presence of responses were reproducibility above 50%, as well as signal-to-noise ratio >3 dB at 1.0 and 1.5 kHz, and >6 dB at 2.0, 3.0, and 4.0 kHz. The protocol was used to allow the analysis of pass/fail criteria for each of the five frequency bands tested in three present frequency bands . The preImmittance testing was performed using an Interacoustics Immittance Meter, model AT235h. The tympanometric curves were classified into: curve A , with a single peak and pressure between −100 and 100 daPa and volume from 0.28 to 2.5 mL; air curve (reduced amplitude) with a reduced compliance peak, below 0.28 mL; curve Ad (increased amplitude) with a double peak, increased compliance above 2.5 mL, and curve B, or flat, without pressure peaks. In a type C curve, the peak shifted to negative pressure below −100 daPa. The acoustic reflex was considered present when present in all frequencies ,25. The For BAEP registration, the Contronic’s Evokadus equipment was used. In individuals with normal hearing: stimulation at high intensities between 75 and 90 dBHL, latency of wave V of 5.5 to 6.0 ms, increasing approximately 2.0 ms until the threshold. The latency of wave I is 1.5 ms, of wave III is around 3.5 ms, and the interpeak intervals are approximately 2.0 ms. At lower intensities, the latencies of all waves increase and wave I can be undetectable. The threshold is defined by the lowest intensity at which the V wave can be replicated . It is aLanguage development was assessed using the Bayley Scales of Infant Development—Third Edition; the version was translated and adapted to Brazilian Portuguese (Bayley III) . The BayParticipants were recruited from the list of live newborns notified by the SINASC (Live Birth Information System)/RESP /SIRAM , provided by the Epidemiological and Environmental Surveillance Department of Juiz de Fora and its surrounding region. All confirmed cases of GZVI were invited to participate through their parents or legal guardians. The first contact was by phone call by one of the team members responsible for the data collection of the study. In the case of fulfilling the eligibility and acceptance criteria regarding participation, a date was scheduled to attend the data collection site . On the day scheduled for the first evaluation, the parents signed an informed consent (IC), and the necessary information was collected for the characterization of the participants and the examinations and treatments carried out until that moment. That same day, Bayley III was also applied in a complete way by a trained team. When realizing that the child was tired, the team interrupted the assessment and scheduled a new date to resume the test application, within a maximum period of seven days. The audiologic exams  and the complementary questionnaire (aiming to survey other risk factors for hearing loss and previous exams) were performed within a maximum period of seven days after the application of the Bayley III in a private clinic specialized in otorhinolaryngology  at no cost to the participants. Parents or guardians were able to monitor all assessments, as long as they did not interfere with the participant’s performance. The evaluations of the participants were programmed with a six-months interval until the age of 48 months, which included a series of complete assessments . Those who attended at least one of these complete assessments were included in the present sample. The majority of participants were older than two years of age upon conduction of the first audiological tests. In each attendance, a follow-up questionnaire was filled out containing information about the treatments and possible complications that occurred during the period, and an appointment was made for the subsequent evaluation. When the participant did not show up at the scheduled date and place, a new telephone contact was made, and the assessment was rescheduled to the closest possible date.All children were submitted to hearing screening with the three scheduled tests in order to observe the results in the tests and an equivalence regarding the involvement or not of the auditory pathways. The child was considered a “pass” in the screening when it showed adequate responses in the three tests. The failure in any of the audiological tests, even with a response in the normal morphological BAEP and a threshold at the minimum level of 30 dBHL, implied referral for differential diagnosis and possible treatments with an otolaryngologist. On the Bayley III, when the CS was below 85 (reduced performance), the participants were referred to speech therapy services. OEAT, immittance testing, BAEP, and Bayley III were repeated every six months.The data were stored and analyzed using the Statistical Package for the Social Sciences (SPSS), version 22.0. To present the sample results, the data were organized into three age groups that had a greater number of attendances in the scheduled evaluations and exams: 30 to 31 months, 36 to 38 months, and 40 to 42 months. The interval between one assessment and another for each participant was at least six months. One of the participants had not yet reached the age of 30 months by the end of the study, and the data from its exams are described separately here and were excluded from statistical analyses of performance in tests and possible associations. Descriptive statistics were performed with values of absolute and relative frequency for categorical variables and mean and standard deviation for continuous variables. For the comparison of two categorical variables, the Chi-square test or the Fisher’s exact test was used. To compare a numeric and a categorical variable, the Mann–Whitney test was used due to the sample size. For all analyses, the significance level of α = 0.05 was considered. The group of participants in this study comprised 15 children. Most (nine) were female and the majority of ZikV infections occurred in the second trimester of pregnancy (six cases) . Only onAs this is a series of cases about medium/long-term outcomes of a poorly investigated health condition, we decided to present the data in detail. Child 15 entered the research at the age of 14 months; in the first collection, they had a delayed language acquisition with a CS of 56 on the Bayley III and auditory alterations: OEAT absent in both ears, impedanciometry was not possible, and the BAEP was morphologically present, but with a hearing threshold of 40 dBNa bilaterally. The second collection of this case was at 18 months. The patient maintained a low performance in the CS language of 47 and similar auditory alterations as found in the previous evaluation, except for the BAEP, for which there was an improvement of 5 dBNa in the threshold, keeping it 35 dBNa below the optimal value. This child was referred to the speech therapy and otorhinolaryngology services since the first exam, and the use of bilateral hearing aids was recommended. However, the parents did not accept the performance of this procedure until the last contact the research team had with them; at that time, the infant was 18 months old. Considering all ages evaluated, eight (53.33%) of the fifteen study participants showed altered results in at least one of the hearing tests and seven participants (46.67%) showed a lower-than-expected performance in language in at least one of the assessments in the study period. Four (26.67%) of them simultaneously presented these negative outcomes, with hearing and language changes at the same evaluated age. The age group of 30 to 31 months (nine children) had the largest number of participants with changes (six or 66.67%) in the tests and examinations performed, four (44.44%) had lower than expected language performance, four (44.44%) had alterations in at least one hearing test, and two (22.22%) presented both outcomes simultaneously. Attention is drawn to the cases of Participants 5 and 9. All their hearing tests were altered, thus suggesting a conductive hearing loss and a low performance on the language scale.The age group of 36 to 38 months had the highest number of participants evaluated (11 children), but proportionally, it had the lowest number of participants with altered results in tests and exams (four or 36.40%): three (27.27%) with alteration in some hearing test; however, there was no language impairment. The age group of 40 to 42 months counted eight children with some altered results in half of them (four); two (25%) showed changes in language acquisition, three (37.50%) showed changes in at least one of the hearing tests, and one (12.50%) showed both outcomes. Participants 5 and 9, who had changes in audiological exams at the first assessment, showed improvement in language development after clinical treatment for serous otitis media, performing within normal limits on the Bayley III in the next assessment (36 to 38 months). Participant 8 performed the audiological exams for the first time at 42 months and showed a similar outcome to that of Participants 5 and 9. There were changes in all tests, suggesting a conductive hearing loss accompanied by language impairment. This participant returned at 47 months, after clinical treatment for serous otitis media with an otorhinolaryngologist. They presented normal results in all audiological exams, but it was not possible to apply the Bayley III due to the age limit allowed for comparison with normative data (42 months). However, the parents reported an improvement in their verbal communication and the researchers noticed a greater participation during data collection. p-value in the age groups of which it was possible to perform the analysis  performed below the expected for the age on the Bayley III in some of the assessments. Considering the studies already published to date, this is the first to report on specific hearing and language outcomes in children older than two years of age in cases where there was ZikV.In the present sample, four (26.7%) of the fifteen participants fell within CZS. participants 1 and 14 had a similar condition, with central changes in the acquisition of language due to an extensive CNS malformation. In the other two cases, CNS changes were minor, but Participant 15 had peripheral sensorineural hearing impairment and a delayed language development; the other participant (#6) did not show changes in audiological or language tests in studied ages. It is not possible to compare the incidence of CZS in confirmed cases of infection during the gestational period due to the lack of studies that used the same inclusion and exclusion criteria as those of the present study (only cases confirmed by laboratory tests), but it is considered high because it affected more than a quarter of the cases.As for the presence of auditory alterations in cases of CZS, the present results are in agreement with the cases reported by Abramov et al. , since tConsidering the sample as a whole, the incidence of conductive hearing disorders was the most prevalent. It occurred in almost half of the participants (46.7%). It is indicative of secretory otitis media (SOM), with the presence of secretion in the middle ear with no signs of acute infection. Considering the age of evaluation of these cases (between 30 to 42 months), the prevalence can be considered high . This ouParticipants 5, 8, and 9 presented similar results, with changes in all audiological exams of a conductive characteristic and impaired language acquisition. These findings reinforce the need to perform tests that allow the diagnosis of alterations of this type at least until the age of three in populations similar as that of the present study, ensuring the possibility of referral to treatments that enable an appropriate language development, which meets the recommendations of the Joint Committee On Infant Hearing (JCIH) . ChildreParticipants who showed changes in OEAT and immittance testing, but without BAEP involvement, had no language acquisition problems, suggesting that language acquisition is more affected when there is a greater involvement of the middle ear and/or neural pathways. It is worth mentioning that there were language alterations in participants who had all audiological exams altered or extensive CNS malformations, except for Participant 12, in whom these conditions were not present, but who had a delay in language acquisition in the first assessment performed at 30/31 months. It is believed that the case of this participant is related to a family component of extreme care or overprotection , since aTo date, no studies have been published on language development and hearing disorders in infants or preschoolers who were exposed to ZikV during pregnancy , making Although there was no significant association between the BAEP results and the Bayley III, it is important to note that in all cases where there was a bilateral alteration in this exam, there was an alteration in language development. This seems to be a good indicator of the risk of delay in language acquisition. In this way, the results of the present study reinforce the idea that that it must be the exam of choice for monitoring cases of GZVI. Its periodic performance is recommended, even with normal BAEP results in the first year of life. The BAEP is the best choice for assessing hearing acuity in children  and is aAs limitations, the fact that not all participants were evaluated in the three age groups stands out, but this is expected due to the longitudinal character of the study. This situation was aggravated by the need to interrupt collections as of March 2020 due to the COVID-19 pandemic. This study design did not enable statistical inferences between hearing disorders and CZVI, but as a strength, it reveals the relevant occurrence of these disorders in the region studied and allows for future studies in older ages of this population, mainly to investigate the first school years.The case series of the present study reveals that infants and preschoolers with CZVI may present early neurosensory loss and late hearing loss, with fluctuating character. Most hearing losses were conductive (46.67%); only one case (6%) showed a sensorineural hearing loss. Even if there were no significant association between the audiological exams results and the Bayley III performance, in the present sample, language development was below expectations for the age in the participants who had alterations in the three audiological exams, when there is an early hearing loss or extensive lesions to the CNS.The results suggest that it is important to perform audiological exams, especially the morphological and auditory threshold BAEP, to monitor cases of CZVI up to the age of three. Prompt referrals for treatments and assessment of language development are recommended in cases when there is a change in the results of these exams.


We report an unprecedented case of a young patient with epilepsy coexisting with acute zonal occult outer retinopathy (AZOOR), a rare white dot syndrome of unknown etiology, associated with damage to the large zones of the outer retina. Recently, it has been established that epileptic episodes contribute to an inflammatory response both in the brain and the retina. A 13-year-old male patient with epilepsy was referred by a neurologist for an ophthalmologic consultation due to a sudden deterioration of visual acuity in the left eye. The examination, with a key role of multimodal imaging including color fundus photography, fluorescein angiography, indocyanine green angiography (ICGA), fundus autofluorescence (FAF), swept-source optical coherence tomography (SS-OCT) with visual field assessment, and electroretinography indicated AZOOR as the underlying entity. Findings at the first admission included enlargement of the blind spot in visual field examination along a typical trizonal pattern, which was revealed by FAF, ICGA, and SS-OCT in the left eye. The right eye exhibited no abnormalities. Seminal follow-up revealed no changes in best corrected visual acuity, and multimodal imaging findings remain unaltered. Thus, no medical intervention is required. Our case and recent laboratory findings suggest a causative link between epilepsy and retinal disorders, although this issue requires further research. Acute zonal occult outer retinopathy (AZOOR) is a rare clinical entity of unclear etiology, which invariably leads to dysfunction of the outer retinal zones. Typically, AZOOR occurs in young women with myopia. Several causative factors have been implicated in the development of this disorder, including infections and autoimmune diseases . The maiEpilepsy is a group of diseases characterized by recurrent episodes of seizures . A recenA 13-year-old male patient with a recent diagnosis of epilepsy was referred by his neurologist to the Department of Ophthalmology and Ocular Oncology at Jagiellonian University Medical College in Kraków, Poland, due to visual deterioration and visual field disturbances in the left eye. The patient denied photopsias. Best corrected visual acuity was 20/20 in the left eye and 20/25 in the right eye (due to anisometropia), while the refractive errors were −2.0/−0.25 axis 173 and −4.25/−0.25 axis 150, respectively. The pupils were equal and exhibited no relative afferent pupillary defect. Intraocular pressure, color vision testing, and slit-lamp biomicroscopy of the anterior segment of the eye indicated no abnormalities, whereas fundus examination revealed only an area of a thinner retina with some hyperpigmentation A. VisualThe patient had previously been treated at a neurological unit where he was diagnosed with epilepsy on the basis of two unprovoked seizures and epileptic activity on electroencephalography. To prevent any further epileptic seizures, valproate (Depakine) had been administered with satisfactory results. The treatment lasted four years and was discontinued without a relapse of seizures.Magnetic resonance imaging of the brain and orbits revealed no abnormalities that could explain the symptoms. Routine blood screening as well as titers of specific antibodies  were within reference ranges. Possible infective causes were excluded.In the left eye, swept-source OCT (SS-OCT) showed peripapillary disruption of the ellipsoid zone (which is associated with photoreceptor damage) E, while Electrophysiological examinations were performed using EP-1000 . All examinations were compliant with the International Society for Clinical Electrophysiology of Vision standards ,6,7. PatThe analysis of the clinical data, with a pivotal role of additional imaging, has led to the diagnosis of AZOOR. As there was no foveal involvement and normal visual acuity was preserved, there were no indications for pharmacological treatment. The patient remains in a semiannual follow-up at our department, lasting over five years since the diagnosis, during which a thorough assessment with color fundus photography, SS-OCT, FAF, and ICGA is performed. During the follow-up, no substantial progression of the disorder has been observed. Findings for the left eye on all diagnostic modalities have remained stable during the follow-up B,D,F,H,JTo the best of our knowledge, this is the first patient with AZOOR and concomitant epilepsy reported in the literature. To date, AZOOR has been associated with numerous diseases including herpes simplex dermatitis, central nervous system abnormalities, viral infections, migraines, tick bites, and autoimmune diseases (in 28% of cases). An association with pregnancy was also reported . CertainAs the results of some imaging modalities can be ambiguous, the ophthalmologic community recommends ERG as a diagnostic tool of choice in patients with suspected AZOOR, as the tracing is always abnormal ,9,16,17.No consensus on the treatment of AZOOR has been reached. However, systemic corticosteroids as well as immunosuppressive, antiviral, and antibacterial drugs are regarded as the main options. The role of topical medications is still discussed, since only vitritis has been reported to resolve once the treatment is started . Some auNumerous studies linked visual disturbances and structural changes of the different retinal layers with antiepileptic drugs, but the effects of seizures themselves were not assessed in detail ,21,22,23Recent investigations have established a direct link between responses in rat retina and epileptic episodes . ResearcIn summary, our case demonstrates the necessity for a thorough and multidisciplinary approach to retinal diseases such as AZOOR, especially in patients with concomitant diseases, such as epilepsy. In the presence of nearsightedness, normal fundus appearance, and the presumed impact of antiepileptic treatment, the presence of AZOOR may be easily overlooked.


Aeq,8h), earplug personal attenuation ratings (PARs), and pure-tone audiometric tests were performed. The average LAeq,8h of noise-exposed workers was 87 dB (A) with a mean PAR of 7 dB. The prevalence of high-frequency hearing loss was 65% for noise-exposed workers and 33% for the non-noise-exposed employees. The use of earplugs had no observable effect on the prevalence of high-frequency hearing loss of the study participants . No significant relationship between the effectiveness offered by earplug use and high-frequency hearing thresholds at 3, 4, and 6 kHz was found . The mandatory requirement of earplug use without individualized training on how to wear HPDs correctly had no detectable effect on the prevention of hearing loss at the auto parts factory. The hearing conservation program at the surveyed factory was not effective. Periodic hearing tests, earplug fit testing, expanding the offer of different types of hearing protection, and employee education about the importance of protecting their hearing were recommended to the occupational health and safety program.A survey was administered to 385 noise-exposed workers from an auto parts factory and 1268 non-noise-exposed health department employees in China. Individual 8 h A-weighted equivalent sound levels (L Noise-induced hearing loss (NIHL) is the third most prevalent chronic physical condition among adults in the United States . In ChinIn China, workers are required to wear HPDs when they are exposed to workplace noise levels above 85 dB (A) over 8 h . The sinSince HPDs are widely used in noisy industrial environments, it is essential to determine whether HPDs are effective in preventing noise-induced hearing loss. This study at an auto parts factory in China was designed to investigate the attenuation offered by earplugs and their effectiveness in preventing NIHL.This study was approved by the Ethics Committee of Jiangsu Provincial Center for Disease Control and Prevention (JSCDC). All participants provided their consent to participate in the study. Information obtained by a questionnaire and company data were used to obtain historical noise exposure information.This case-control study was conducted at an auto parts factory and the Jiangsu Provincial health department in eastern China. JSCDC conducted an initial investigation for participants routinely exposed to occupational noise at an auto parts factory in August 2018. A second investigation for participants who were not exposed to occupational noise was conducted from December 2018 to September 2019. The non-noise-exposed workers were employees from the Jiangsu Provincial health department, including two clinical hospitals and 10 local centers for Disease Control and Prevention. Each participant was given an otoscopic exam, tympanometry, and pure-tone audiometry (500–8000 Hz). The inclusion criteria were (1) no history of prior employment in a high noise environment, (2) no military service history or shooting activities, (3) a clear view of the tympanic membrane on otoscopic examination, and (4) type A tympanogram. To be eligible for participation, noise-exposed workers had to have a minimum of one year of employment at their current job. A total of 385 noise-exposed workers and 1268 non-noise-exposed employees met the eligibility criteria from original pools of 504 and 1672, respectively.The production area of the surveyed auto parts factory consisted of two separated one-story buildings. Each building had multiple production lines and eight job titles . Workers were not exposed to ototoxicants at this factory.There were no physical barriers/walls between lines. The factory operated three rotating 8 h shifts, 5 days a week. Overtime work was available but not mandatory. The auto parts factory had a full-time health and safety manager who provided safety and health training and organized occupational health hazard monitoring and health surveillance. Part-time safety supervisors in each production area on each work shift assisted the safety manager. They were responsible for the field safety and occupational issues of the specific production area.Noise-exposed workers were offered earplugs for voluntary use when the company started operations in 2003. In 2012, the use of 3M 1270 premolded earplugs became mandatory during workers’ full work shifts. It was the only type and model of earplug provided to the workers since then. The labeled attenuation value (SNR) for this earplug is 25 dB. All workers received pre-employment training and annual refresher training thereafter. The pre-employment training included environmental, safety, and health knowledge. The annual refresher consisted of group presentations including how to wear earplugs correctly with a written examination afterward.Each participant was required to complete a health questionnaire, which was administered through a face-to-face interview. The following information was collected: demographic information , noise exposure history , and history of ear diseases and use of ototoxic drugs. Noise-exposed employees were required to provide their HPD use history.Aeq,8h) data were extracted from the dosimeters directly, regardless of the length of the shift.Area full-shift noise measurements were conducted at the worksite of the participants who were not exposed to noise using dBadge noise dosimeters . Each dosimeter was calibrated before and after the measurement using a Casella CEL-120/2 Sound Level Calibrator. The noise measurement protocol was consistent with NIOSH recommendations for workplace noise exposure sampling . The equAeq,8h and kurtosis calculation. To address the non-Gaussian noise in this study, the kurtosis of the recorded noise signal was computed for consecutive 40 s time windows without overlap over the full shift using MATLAB software [Each noise-exposed worker from the auto parts factory wore an ASV5910-R digital recorder , operating continuously with 16-bit resolution at a 48 kHz sampling rate, during their 8 h shifts. This instrument was selected because of our interest in the temporal characteristics of the noise exposure. The recorders comply with IEC 61672 and IEC 61252 standards and were used in previous studies . The recsoftware . The measoftware .Aeq,8h for noise-exposed workers was calculated based on the individual full-shift noise recording data using a program written with MATLAB  software. This protocol was consistent with NIOSH recommendations for workplace noise exposure sampling [Aeq,8h  isThe Lsampling . BecauseAeq,8h and service years, was used to quantify the cumulative noise exposure for each participant [The cumulative noise exposure (CNE), a composite noise exposure index based on Lticipant . The CNE1 is the years of service before 2012 and T2 is the years of service after 2012. For example, if a worker had been working 9 years, the ACNE was calculated by LAeq,8h with T1 for the first 3 years and the ALAeq,8h with T2 for the last 6 years (keeping in mind that the investigation was conducted in 2018). If a worker started the job after 2012, then T1 = 0, and the ACNE was calculated by the ALAeq,8h for his work; T2 was the number of work years.Considering that this company only started its hearing protection program in 2012, the CNE for workers who started working before 2012 needed to be adjusted. The adjusted CNE (ACNE) was defined asThe field attenuation estimation system used in this study was the 3M E-A-Rfit Dual-Ear Validation System , which iAeq,8h).Fit testing was performed in a conference room at the local hospital. In 2018, 6 years after earplug use became mandatory, workers’ PARs were measured by asking the workers to fit the surrogate probed test earplugs by themselves the way they normally wore them during their work shift. No coaching or demonstration was provided. The PAR passing criterion was defined as being greater than the target minimum attenuation, which was based on the individual noise exposure results see . This waAll participants underwent pure-tone audiometry testing. Audiometry was performed in a double-walled, sound-treated booth at a local hospital or in a single-walled audiometric booth in the JSCDC mobile vehicle. A certified audiologist conducted the audiometric testing using a calibrated audiometer . Air-conduction pure-tone hearing threshold (HT) levels were tested from 500 to 8000 Hz in each ear. For noise-exposed workers, hearing testing was conducted at least 16 h after the workers’ last occupational noise exposure. High-frequency hearing thresholds (HFHTs) used in the analysis were defined as the average thresholds at 3, 4, and 6 kHz in the better ear. The better ear was used because this was the criterion for establishing median hearing threshold levels of the control group [Descriptive statistics of information from questionnaires, noise measurements, PARs, and the prevalence of high-frequency hearing loss of each age group were calculated using Microsoft Excel. Because SNR is intended to be subtracted from the C-weighted sound level, while PAR is subtracted from the A-weighted sound level, 2 dB C minus A corrected SNR  was applp < 0.05 was adopted for all analyses. The correlation between PAR and LAeq,8h was examined with the Pearson correlation coefficient. A one-way analysis of variance (ANOVA) was conducted to evaluate the difference of mean PAR by LAeq,8h group. To analyze the distribution characteristic of HTs at each frequency for all participants, a general linear model (GLM) was conducted, followed by Tukey’s studentized range (HSD) test.Statistical analyses were performed with SAS . A statistical significance level of Aeq,8h, and service years), the estimated median high-frequency hearing threshold for each noise-exposed worker was calculated. The comparison of hearing thresholds between noise-exposed workers, non-noise-exposed employees, and ISO-estimated hearing threshold was conducted exclusively within gender by age group. The hearing threshold comparison was conducted by a GLM procedure following HSD test.Based on International Organization for Standardization (ISO)1999:2013  and alcohol consumption  were not confounders using a generalized linear model, a final linear model “log (HFHT) = a + b (CNE) + c (CNE-ACNE) + age + gender” was conducted to check the association between HFHT with CNE, earplug protection, age, and gender. Log (HFHT) was used because the HFHT was not normally distributed.Two analyses were carried out to evaluate a possible association between earplug use and hearing outcome. First, correlations between the prevalence of high-frequency hearing loss and CNE, ACNE, earplug attenuation, age, and gender were examined by Pearson correlation analysis. Second, after confirming that smoking . For non-noise-exposed employees, 36% were male and 64% were female; their median tenure was 11 years (25th–75th percentile: 5–23 years); their mean age was 38 years with an SD of 11 years. The male employees (41 ± 10.6 years) were older than the female employees (37 ± 10.2 years) .A total of 385 noise-exposed workers and 1268 non-noise-exposed employees participated in this study. Among noise-exposed workers, 70% were male and 30% were female; their median duration of noise exposure was 8 years (25th–75th percentile: 4–11 years); their median age was 37 years with a standard deviation (SD) of 7 years. The male workers (35 ± 7.5 years) were younger than the female workers (40 ± 5.5 years) . Noise exposures ranged from 77 to 102 dB (A) with an SD of 3.5 dB (A), with a mean kurtosis of 19.8 and an SD of 12.6.The area full-shift noise levels at non-noise-exposed employee workstations ranged from 65 to 78 dB (A), while the average LAeq,8h estimates for 78.2% of the noise-exposed workers (n = 301) exceeded 85 dB (A). The highest level of noise was generated by the tube cutting, grinding, and machining processes. n = 143) of the workers had LAeq,8h between 85 and 87 dB (A), and 33.2% (n = 128) were exposed to levels that ranged from 88 to 93 dB (A). The median CNE level for all noise-exposed workers was 95 dB (A)·year, which ranged from 80 dB (A)·year to as much as 111 dB (A)·year.The Ln = 149) of the workers. The SNR for this specific earplug is 25 dB. To compare these obtained PARs to the labeled attenuation values, a minus 2 dB correction was applied to the SNR [ the SNR . Thus, tn = 80) of the noise-exposed workers’ PARs achieved the de-rated SNR, while only one worker’s PAR achieved the SNR. The PAR passing rate was 53.7% among 315 workers whose noise exposure levels were 85 dB (A) or above.In China, a 40% de-rating of the SNR is required in the workplace, which reduces the expected attenuation from this specific plug to 15 dB. The recorded mean PAR  was well below the SNR and de-rated SNR (15 dB). Twenty-one percent . This indicates a slight negative linear relationship between PAR and LAeq,8h, but it did not reach significance. Analysis of variance (ANOVA) testing shows that there was no difference in the mean PAR among LAeq.8h groups , as shown in p = 0.038).The Pearson correlation between PAR and Lp = 0.259). Male workers had worse hearing than female workers , except in the 50–59 age group . We observed no significant difference between left and right ears . Male workers with CNE ≥ 94 dB (A)·year had significant higher average thresholds at each frequency (in the better ear) than workers with CNE < 94 dB (A)·year . A similar observation was found for female workers  at 1000 (p = 0.028), 2000 (p = 0.030), and 6000 Hz (p = 0.008). Moreover, a significant difference of hearing threshold was found between male and female workers with CNE ≥ 94 dB (A)·year  at 500 (p = 0.041), 3000 (p = 0.025), and 4000 Hz (p = 0.002)  .p < 0.001) at each frequency except 4000 Hz (p = 0.848). Male noise-exposed workers had worse hearing threshold than non-noise-exposed male workers  at each frequency, which was similar for female workers  from 500 to 4000 Hz.ISO-estimated median hearing thresholds (HT) were significantly better than actual thresholds of noise-exposed workers in this study  had audiometric configurations showing characteristics of a high-frequency hearing loss, a percentage significantly higher than that of non-noise-exposed study participants within gender (Sixty-five percent of the noise-exposed workers (n gender .p < 0.001), while there was no difference between female and male workers (p = 0.995). Because the hearing thresholds used here were not adjusted by age and gender, logistic regression was conducted to evaluate their contribution to the hearing outcome. The results indicated that the prevalence of high-frequency hearing loss was associated with age . Males had a higher prevalence of high-frequency hearing loss than females . The prevalence of high-frequency hearing loss increased slightly with the CNE  but not significantly. The use of earplugs had no observable effect on the prevalence of high-frequency hearing loss .The Pearson correlation test showed that the prevalence of high-frequency hearing loss increased significantly with the increase in CNE, ACNE, and age (p = 0.002), females had lower hearing thresholds than males , and HFHTs increased by age . There was no significant association between PARs and hearing thresholds .The median HFHT at 3, 4, and 6 kHz in the better ear was 15 dB HL . An analysis with a linear model found that HFHTs significantly increased with the increase in CNE levels  with 315 workers being exposed to noise level 85 dB (A) or above. The mean PAR offered by the earplugs was 7 dB, which could theoretically protect 53.7% of the 315 noise-exposed workers by reducing in-the-ear noise exposure to less than 85 dB (A).The objective of this investigation was to examine earplug attenuation and its effectiveness in prevention of hearing loss among auto parts factory workers. The average LMany occupational and non-occupational factors can have an impact on hearing thresholds. In this investigation, we controlled for other work-related factors  and examined the contribution of other factors such as age, gender, smoking, and alcohol consumption. The prevalence of high-frequency hearing loss among the noise-exposed workers was 67%, which was higher than the 33% of non-noise-exposed study participants with a hearing loss. The difference in hearing thresholds between noise-exposed workers and non-noise-exposed employees at each frequency within both male and female increased with worker age, especially at 4, 6, and 8 kHz, for both genders. This indicates that occupational noise exposure plays a significant effect on the hearing of the noise-exposed workers.Aeq,8h for the duration of exposure (in work years) was used to estimate noise-exposure history. To investigate the effectiveness of HPDs based on the personal attenuation rating, the ALAeq,8h was calculated from the LAeq,8h minus the individual’s PAR, and then the ACNE was calculated from the ALAeq,8h and the exposure duration in years. If earplugs provided enough attenuation for the individual worker, both the prevalence of high-frequency hearing loss and HFHT at a level of CNE should be higher (worse hearing) than that at the same level of ACNE. To test this hypothesis, the difference between CNE and ACNE at the same level was used as an indicator of earplug effectiveness.To determine whether the use of earplugs effectively protected workers from noise-induced hearing loss, two outcomes were examined: high-frequency hearing loss and HFHTs. A CNE metric that combines LBoth the generalized linear model and logistic regression model showed that earplug attenuation had no significant effect on the hearing of participants. These findings are in agreement with the evidence from previous studies that the use of HPDs in the workplace is often inconsistent and not fully effective ,20,21,22There are many possible explanations for workers not achieving the desired attenuation. Poor fitting techniques  were observed by the research staff. When the researcher attached the dual-element microphone array to the probed test earplugs, the researcher could often feel that the earplugs did not seal these workers’ ear canals. A wide range of PAR values from 0 to 25 dB was observed among workers who improperly fit their earplugs. More than half of the workers achieved 5 dB or lower attenuation. These results, however, must be interpreted with caution. Workers might have worn earplugs differently or even fit them better than they normally do because they realized that they were being tested. Therefore, it is conceivable that the amount of attenuation the workers received on a typical workday is different from what was obtained in this study. Still, they are a robust illustration of the challenges in achieving the desired earplug attenuation.Some workers were observed not wearing earplugs all the time during their full shift even though they were required to do so. Some workers took their earplugs off when they needed to communicate. Similar observations were made by Nélisse , who tesIn addition, only one type of premolded earplug was offered to the workers in the 6-year period that preceded this study. A previous survey conducted by the JSCDC for 503 noise-exposed workers at nine manufacturing facilities in China revealed that 10% of the workers were unable to achieve target attenuation from the assigned HPD after individual training. For those who needed and were provided a different product or size of HPD, 70% were able to achieve the target attenuation . SimilarFinally, the group training method may have contributed to the lack of effectiveness of earplugs observed in this study. The surveyed auto parts factory had a well-organized occupational health and safety program, in which workers received a group pre-employment training and annual refresher training followed by a written exam. However, multiple studies of the training methods have shown that they failed to teach how to properly fit their earplugs ,19,20,21In summary, despite being required for almost a decade, as worn by this group of workers, earplugs generally did not provide the needed attenuation and consequently showed no significant protective effect on the hearing of the participants. Similar findings have been reported earlier ,4,5. EveAeq,8h. This finding was different from the findings of Chen et al. [Additional findings from this study provide insight that could prove valuable in increasing the effectiveness of hearing protection programs. First, no PAR increment was detected with increasing Ln et al. . They reSecond, this study found that the ISO-estimated median HTs were significantly better than the real-world thresholds of noise-exposed workers in this study. Differences in the population characteristics and noise exposure estimations might explain this finding. The study was able to conduct noise measurements for all participants and incorporate kurtosis in the calculation of a cumulative noise estimate. Kurtosis has been reported to play an important role in evaluating NIHL ,14 and ep = 0.995). In this study, the average age of male noise-exposed workers was 5 years younger than female noise-exposed workers. Examining age and gender through a logistic regression model and in a generalized linear model, age was shown to have a significant effect on the noise-exposed workers’ hearing. This finding is consistent with the reports from Groenewold et al. [Third, the Pearson correlation test showed no difference in the hearing outcomes between female and male noise-exposed workers p = 0.99. In thisd et al. . It is uThe case-control design and having only one instance of each of the measurements  are the important limitations of this study. Of all information collected, noise exposures were the most robust of the studied variables. We were unable to use historical audiometric data from the noise-exposed workers, as companies are only required to do a periodic pass/fail hearing screening. In addition, we only conducted fit testing once. A single PAR result cannot sufficiently represent the attenuation obtained throughout the years, perhaps not even in a day. Studies with repeated measurements could provide more robust evidence on the real-world attenuation provided by the HPDs. Although the workers were exposed to eight different types of work noise, the generality of the findings in this study may be limited by the fact that the data came from a single factory. Similar studies in different industries will be helpful to further evaluate the actual protection capability of HPDs in the real world.Despite these limitations, we observed an association between noise exposure and the hearing of this population of relatively young workers and found no evidence of any significant contribution from the existing hearing protection program. In current practice, the use of earplugs had limited effectiveness in the prevention of hearing loss at the auto parts factory. In addition, a single choice of HPDs for the workers could be a barrier to an effective hearing loss prevention program. Our study suggests that several programmatic elements can impact the success of hearing loss prevention initiatives. Periodic hearing tests, earplug fit testing, expanding the offer of different types of hearing protection, and employee education about the importance of protecting their hearing were recommended to the surveyed factory’s occupational health and safety program.


The five avian influenza A/H9N2 viruses isolated from wild birds in Jiangxi, China in 2015 are novel reassortants which most likely evolved from multiple lineages. They shared a high similarity with isolates from poultry, suggesting a frequent contact and continuous viral circulation at the bird‐poultry interface. Given the continuous reassortment of H9N2 viruses, it will of substantial importance to implement routine surveillance in wild birds to successfully control avian influenza viruses and better the early warning system of the emerging reassortants with pandemic potential. Avian influenza A virus (H9N2) has become widespread and established a stable lineage in domestic poultry in China since its first identification in Guangdong Province in 1994  and subsequently shipped to the laboratory and stored frozen at −80℃. Virus isolation was conducted in 9‐ to 11‐day‐old specific pathogen‐free (SPF) embryonated chicken eggs. Five out of 45 samples had haemagglutination activity. The viral RNAs of these positive samples were extracted from allantoic fluid with haemagglutination activity using RNeasy Mini Kit . The superscript III reverse transcription‐PCR (RT‐PCR) kit (Invitrogen) was used for the reverse transcription synthesis viral cDNA. All segments were amplified using a Phusion high‐Fidelity PCR system (New England Biolabs) adhering to the manufactory guide. Full‐genome sequencing was performed with Applied Biosystems Automated 3730xl DNA Analyzer. subtype. All animal works are in according with the Animal Care and Use Committee guidelines of China Agricultural University (SKLAB‐B‐2010‐003).To investigate the phylogenetic characterization of H9N2 viruses, phylogenetic trees of eight genes were constructed using the maximum likelihood method based on the Kimura 2‐parameter model Kimura,  in MEGA Ardeola bacchus), Yellow Bittern (Ixobrychus sinensis) and Striated heron (Butorides striatus). They are designated as A/Chinese Pond Heron/Jiangxi/K64/2015(H9N2), A/Yellow Bittern/Jiangxi/K65/2015(H9N2), A/Striated Heron/Jiangxi/K66/2015(H9N2), A/Chinese Pond Heron/Jiangxi/K76/2015(H9N2) and A/Yellow Bittern/Jiangxi/K77/2015(H9N2). Phylogenetic analyses showed that all five isolates belonged to the H9N2 subtype. Notably, there is a distinct phylogenetic structure in each gene segment, with lineages derived from H9N2 and H5N1 . H9N2 positive samples were collected from Chinese Pond Heron Ardeola acchus, Y1 Figure . Amongst1 Figure . This ph1 Figure . The abi1 Figure  highlighTo address the genetic similarity between viruses isolated from wild birds and domestic poultry, average genetic distance, defined as the number of base substitutions per site from averaging over all sequence pairs, was used to represent the evolutionary divergence between two species. Analysis were calculated using the Kimura 2‐parameter substitution model with a gamma‐distributed variation rate among sites (shape parameter = 4).The average genetic distance between domestic poultry and wild birds vary from 0.001 to 0.105 substitutions per site depending on the segment  with the accession numbers of EPI1485062‐EPI1485079 and EPI1485083‐EPI1485104.Sequence data have been deposited into the GISAID platform (FigS1‐TableS1Click here for additional data file.


Background: Triclosan (TCS) is a widely used antibacterial agent in personal care products and is ubiquitous in the environment. We aimed to examine whether TCS exposure affects microbiota in the gastrointestinal tract of zebrafish.Methods: After exposure to TCS 0 , 0.03, 0.3, 3, 30, 100, and 300ng/ml, respectively, from day 0 to 120days post fertilization (dpf), or for 7days in adult 4-month zebrafish, the long- and short-term impact of TCS exposure on the microbiome in the gastrointestinal tract was evaluated by analyzing 16S rRNA gene V3-V4 region sequencing.Results: The top two most dominant microbiota phyla were Proteobacteria and Fusobacteria phylum in all zebrafish groups. In TCS exposure 0–120 dpf, compared with DMSO control, the mean number of microbial operational taxonomic units (OTUs) was 54.46 lower (p<0.0001), Chao indice 41.40 lower (p=0.0004), and Ace indice 34.10 lower (p=0.0044) in TCS 300ng/ml group, but no change was observed in most of the other TCS concentrations. PCoA diagram showed that the microbial community in the long-term TCS 300ng/ml exposure group clustered differently from those in the DMSO control and other TCS exposure groups. A shorter body length of the zebrafish was observed in the long-term TCS exposure at 0.03, 100, and 300ng/ml. For 7-day short-term exposure in adult zebrafish, no difference was observed in alpha or beta diversity of microbiota nor the relative abundance of Proteobacteria or Fusobacteria phylum among DMSO control and any TCS levels, but a minor difference in microbial composition was observed for TCS exposure.Conclusions: Long-term exposure to high TCS concentration in a window from early embryonic life to early adulthood may reduce diversity and alter the composition of microbiota in the gastrointestinal tract. The effect of short-term TCS exposure was not observed on the diversity of microbiota but there was a minor change of microbial composition in adult zebrafish with TCS exposure. It inhiynthesis . TCS is ynthesis . This maynthesis . TCS hasynthesis . The medynthesis . Despiteynthesis .Early life is a period critical for the initiation and development of gut microbiota, which is known to be related to a variety of health outcomes . PreviouZebrafish is a well-established aquatic animal model with well-known ecological dynamics of gut microbial communities , 2016. ITriclosan , purity ≥97.0%) and Dimethyl Sulfoxide  were purchased from Sigma-Aldrich .Danio rerio) were used in this study. Male and female zebrafish aged 5-months-old were acclimated in glass tanks containing regular fish-raising water (without TCS) under a photoperiod of 14:10h light/dark cycle for 4weeks. Zebrafish were fed with brine shrimp twice per day at 09:00 and 18:00h. At 30min after feeding, the remaining food and feces were removed.The zebrafish wild type strain AB . Unfertilized eggs were discarded. Zebrafish larvae were fed with paramecium twice per day from 3 dpf till 14 dpf. After that, zebrafish larvae were fed with brine shrimp from 15 dpf. The pH was maintained at 7.5±0.5, temperature maintained at 28±0.5°C, conductivity maintained at 550±50 μS, and dissolved oxygen of the exposure media were monitored.The exposure doses were set at 9 levels of TCS exposure from 0 , 0.03, 0.3, 3, 30, 100, 300 to 900ng/ml, with consideration of the exposure levels of human, fish and the aquatic environment . ConcentTo examine the effect of long-term exposure from early life, TCS exposure was started in fertilized zebrafish eggs. Fertilized zebrafish eggs were collected and cultured at 28.0±0.5°C under 14:10 light/dark photoperiod cycle in a climate chamber. We collected fertilized eggs within 30min post fertilization and randomly assigned 270 fertilized zebrafish eggs to the 9 TCS exposure groups (30 eggs per TCS exposure group) and raised to 120 dpf. Specifically, we put 30 fertilized zebrafish eggs randomly in each culture dish with 100ml medium. At 14 dpf, zebrafish were transferred from dishes into tanks and continued to culture with 1,000ml exposure medium up to 120 dpf. We changed half of the medium each time twice per day. All glass containers were autoclaved prior to use and all fish tanks were placed in the same room to assume similar environmental conditions.After exposure to TCS/or control, respectively, for 120 dpf, the fish were euthanized by ice-cold water bath immersion on the 120th dpf morning before feeding. The surface of each fish was sterilized using 75% ethanol and then the gastrointestinal tract  to the same 9 TCS exposures levels  as long-term groups for 7days. The gastrointestinal tract samples were also collected with the same sterile method as the long-term exposure groups.In the long-term exposure study, we measured and recorded the body weight and length of zebrafish at 120 dpf. After the zebrafish were euthanized by ice water, dry weight and the length from the anterior mouth to the tail tip were measured.Microbial DNA was extracted from the gastrointestinal tract samples of zebrafish using the beads-beating method as previously described . A detaiThroughout the experimental process , nuclease-free materials were used and gloves were changed between handling each sample to prevent accidental cross-contamination between samples. The background negative control was set and subjected to the same DNA extraction procedure and analysis protocol alongside the intestine samples of zebrafish. The lab technicians were blinded to the exposure status of the zebrafish.High-quality sequence alignments, sequence clustering, and operational taxonomic unit (OTU) delineation were performed as previously described . The QuaThe relative abundances of bacteria were calculated at the levels from phylum to genus. Alpha-diversity indices were calculated and compared between the DMSO control group and exposure groups using linear mixed models. Beta-diversity was evaluated by Principal Coordinate Analysis (PCoA) based on bray-curtis . Sample p<0. 05 were considered statistically significant.To account for observation correlation within the same TCS exposure tank, linear mixed models were used to evaluate the associations of TCS exposure levels with the diversity and relative abundance of microbiota at phylum and genus levels, and with body weight and length of zebrafish. Statistical analysis was carried out in SAS 9.3 . Two-sided values of p=0.988), However, the incidence density of larvae death was higher with higher TCS exposure levels in the first 5days .The number and proportion of male was 4 (50%) in the DMSO control, 2 (28.6%) in the blank control, and 3 (50%), 3 (42.9%), 5 (71.4%), 5 (62.5%), 4 (50%), 3 (42.9%) in TCS 0.03, 0.3, 3, 30, 100, 300ng/ml groups, respectively. There was no statistical difference in male proportion (or sex ratio) among the DMSO control group and other TCS groups. As shown by Kaplan–Meier survival curves , overallp<0.0001), Chao indice 41.40 lower , and Ace indice 34.10 lower  in TCS 300ng/ml group than the DMSO control group (p=0.40) and Simpson indice (p=0.40) in TCS 300ng/ml group were not significantly different from those in DMSO control group.All of the zebrafish at the highest (900ng/ml) TCS group died before 5 dpf, so we did not measure their microbiota. After 120days of TCS exposure, mean (± SE) number of OTUs were 110.00±8.43 in the blank control, 125.75±2.39 in DMSO control, and 96.83±7.91, 101.57±5.13, 126.86±7.43, 110.63±8.45, 123.25±6.49, and 71.29±11.22 in TCS 0.03ng/ml, 0.3ng/ml, 3ng/ml, 30ng/ml, 100ng/ml and 300ng/ml exposure groups, respectively. Linear mixed-model analyses revealed that the mean number of OTUs was 54.46 lower , Fusobacteria , CKC4 , Firmicutes , Bacteroidetes , Planctomycetes , Actinobacteria , Cyanobacteria , Cetobacterium , CKC4_norank , Plesiomonas , Sphingomonas , Aeromonas , Hyphomicrobium , Staphylococcus  than the DMSO control group , but no difference was found in other TCS exposure levels . In the replicate experiment, a shorter body length of zebrafish was observed in long-term TCS exposure at 300ng/ml compared to that of the DMSO control group .In addition, we conducted a replicate experiment to compare the effect between long-term exposure to TCS 300ng/ml and DMSO control starting from the time of fertilized zebrafish eggs to adulthood (75 dpf). The alpha-diversity (number of observed OTU) was lower for gastrointestinal microbiota of zebrafishes exposed to TCS 300ng/ml, and the beta-diversity of microbiota differed between the TCS 300ng/ml and DMSO control groups , S6. TheProteobacteria, the disappearance of Cyanobacteria, and enrichment in CKC4 at the phylum level, and the body length of zebrafish was slightly shorter. We also found that the diversity of microbiota in zebrafish adults with short-term exposure was not affected by any TCS, but a minor change in microbial composition was observed in high TCS exposure.In this study, we explored the effects of long-term exposure to TCS, during the first 120days of life, on microbiota in the gastrointestinal tract using a zebrafish model. The original contribution of this study is the finding that the TCS exposure window spanned the entire period from early embryonic life to early adulthood. We found that long-term exposure to high TCS concentration (300ng/ml) reduced alpha-diversity, and altered the composition of microbiota, with a decrease in Our study found that TCS exposure was associated with decreased richness and alpha-diversity, and altered relative abundance of microbiota phylum in the gastrointestinal tract of zebrafish. Our results were consistent with the only one available study focused on the effects of short-term TCS exposure on the gut microbiota of zebrafish until now , it showThe relationship between the toxic effects of TCS and the induced structural disturbance of intestinal flora has not been demonstrated. After TCS enters the body, it is rapidly removed through the kidney or gastrointestinal tract by metabolizing water-soluble conjugates . While in the colon tissues, the beta-glucuronidase- and sulfatase-expressing gut bacteria could catalyze the hydrolysis of TCS glucuronide and TCS sulfate, and regenerate the TCS . TherefoDeltaproteobacteria in the TCS group only in adolescence, but not in adulthood  was stable in nature or laboratory environment though the difference in bred and raised conditions  and estrogen (E2) had significantly increased abundance of the phylum CKC4  altered the microbiome composition of zebrafish. These associations were not observed in lower TCS concentration exposure, the level commonly detected in rivers and lakes . HoweverE. coli  and four genera  between the DMSO control and blank control groups. It is unknown whether DMSO may modify the association between TCS and the relative abundance of the above microbiota phyla and genera, caution should be paid in interpreting these results.Because TCS has low water solubility, DMSO is commonly added to dissolve TCS in animal studies . PreviouE. coli . In our We found that the body length of fish was shorter in TCS 100 and 300ng/ml exposure. This is consistent with previous studies. It was found that high TCS exposure (600ng/ml) decreases body length in zebrafish embryos . The chaProteobacteria, Fusobacteria, and Firmicutes  of exposure medium per tank, and we changed half of the medium each time, twice per day. All glass tanks/containers were autoclaved prior to use. Thus the water environment was comparable between the tanks except for TCS levels. In the present study, the main finding was the lower microbial diversity in high TCS (300ng/ml) long-term exposure. Due to the antibacterial feature of TCS, this finding is biologically plausible. Consistent with previous research, this study found that the core gastrointestinal microbiota included bacterial phyla rmicutes . The comrmicutes . HoweverIn this study, we found that long-term exposure to high TCS concentration from early embryonic life to early adulthood may lower diversity and alter the composition of microbiota in the gastrointestinal tract of zebrafish. In adult zebrafish with short-term TCS exposure, no effect was observed on the diversity of microbiota in any TCS exposure, but a minor difference in microbial composition was observed in TCS exposure.https://www.ncbi.nlm.nih.gov/PRJNA682251, and https://www.ncbi.nlm.nih.gov/PRJNA759445.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The animal study was reviewed and approved by Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (NO. XHEC-F-2018-059).FO: conceptualization, design, data analysis and interpretation, writing - original draft and revision, and funding acquisition. NT: research conduction, data analysis, and writing - paper draft. PF: research conduction, data analysis and writing -revision. XY: research conduction. YT, RM, and WW: writing -review and revision. FO is the guarantor of this work and had full access to all data. All authors approved the submitted manuscript.This work was supported by grants from the National Natural Science Foundation of China , the Ministry of Science and Technology of China (No. 2017YFE0124700), Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant (20152518), and Shanghai Municipal Health Commission .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hyperglycemia or diabetes mellitus (DM) is a disorder of the endocrine system. In this condition, the body is insulin-deficient or resistant to insulin. Due to insulin deficiency or resistance, the body is unable to process sugar. The worldwide prevalence of diabetes mellitus is rising substantially.Hyperglycemia makes the immune system weak, which increases the risk of infection in a diabetic patient. Fungal infection is more common in DM. Mucormycosis is a rare fungal infection in a healthy individual, but in DM, it can cause severe complications and even be fatal if not treated adequately and timely. In our literature review, a total of 19 published articles from the PubMed database and Google Scholar were included. We combed the PubMed database and Google Scholar by using various inclusion and exclusion criteria. The result of the review study shows the increased risk of mucormycosis in a diabetic patient. Diabetes mellitus (DM) is one of the most common endocrine diseases affecting millions of people worldwide. Cases of DM have risen by more than 300 million in the last 35 years. Its prevalence is increasing rapidly .Diabetes mellitus is the leading cause of kidney dysfunction, blindness, strokes, lower limb amputation, and heart attacks. According to the World Health Organization (WHO), diabetes was the cause of 1.5 million deaths worldwide in 2019, making it the top 10 leading cause of death globally. Common symptoms of DM are frequent urination, increased thirst, hunger, fatigue, blurred vision, and numbness and tingling in the hands and feet [Type 1 DM is due to the complete absence of insulin production in the body because of autoantibodies targeted against pancreatic beta cells . InsulinCommon factors which help in preventing and delaying the onset of DM are regular physical activity, eating healthy, and maintaining average body weight. Routine screening, proper diet, participating in physical activity, and medications help treat and prevent DM complications .Mucormycosis is also called a black fungus. Cases of mucormycosis are infrequent but can be disastrous in patients suffering from immunocompromised conditions such as uncontrolled DM, coronavirus disease 2019 (COVID-19), and hematological cancers [Mucor and Rhizopus are the joint offending agents and spread their infection mainly through inhalation of spores, but different routes are traumatic inoculation and ingestion . Its spoMethodsUsing the PubMed and Google Scholar databases, an in-depth systematic review was performed as per Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .Data Source and StrategyWe used keywords and the Medical Subject Heading (MeSH) strategy to explore and maximize the number of articles from PubMed and Google Scholar.Following keywords are used for the search in Google Scholar as shown in Table We used the following MeSH strategy to search for relevant papers:  AND  AND Mucormycosis OR Rhizopus OR Mucor OR .Inclusion and Exclusion CriteriaThe inclusion criteria include articles published in English in the last 10 years (2011-2021), focusing on all age groups. Study types are used without any restriction, and all kinds of articles like clinical trials, randomized controlled trials, systematic reviews, traditional reviews, and case reports were explored. Restrictions such as age, ethnicity, and demographics were not taken into consideration during the review process. The exclusion criteria included publications whose abstracts were unavailable, incomplete articles, unpublished articles, and articles published before 2011.Screening and Quality/Bias AssessmentWe assessed 35 studies for quality using standardized quality assessment tools. The following methods were used: (1) randomized controlled trials (RCT): Cochrane Risk Assessment tool; (2) systematic reviews: Assessment of Multiple Systematic Reviews (AMSTAR) checklist or PRISMA checklist; (3) non-RCT and observational studies: Newcastle-Ottawa Scale; (4) case reports: Joanna Briggs (JB) check tool; and (5) any research paper without properly defined methods section: Scale for the Assessment of Narrative Review Articles (SANRA) checklist.ResultsWe identified articles using keywords and MeSH strategy for PubMed and Google Scholar databases. Using the MeSH strategy, we yield 3,064 articles from the PubMed database. Due to duplication and irrelevance, we excluded 2,300 articles from PubMed. We were left with 764 articles from PubMed before screening. We excluded 749 articles from the PubMed database based on inclusion-exclusion criteria, poor quality, and unavailability of free text. We selected 15 articles from the PubMed database based on free full text and relevance. To compensate for the excluded articles, we used the Google Scholar database. Keywords used for the Google Scholar database are diabetes mellitus, hyperglycemia, and mucormycosis. We selected four articles based on our inclusion-exclusion criteria from the Google Scholar database. Therefore, finally, 19 articles were selected for systematic review. PRISMA flow diagram is shown in Figure DiscussionPathophysiology of Diabetes MellitusIn type 1 DM, autoantibodies are targeted against beta islets of Langerhans in the pancreas. The normal function of a beta cell is to produce insulin, which helps regulate blood glucose levels in the body . An antiType 2 DM is due to inadequate action of insulin and a decrease in the production of insulin by beta cells. Reduced sensitivity of cells to insulin leads to the development of insulin resistance . To compDiabetes and Immune SystemPersistent high glucose levels in the blood interfere with the normal functioning of mitochondria and stimulate the production of a toxic molecule known as reactive oxygen species (ROS) . The forInnate immunity and adaptive immunity comprise the immune system of the human body. Neutrophils, natural killer (NK) cells, and dendritic cells play a crucial role in innate immunity, whereas B lymphocytes and T lymphocytes are part of adaptive immunity . DM impaThe immune system neutralizes Mucorales by chemotaxis, phagocytosis, and intracellular killing via the oxidative and non-oxidative mechanisms . For theIron is a significant element in the development and cell growth of humans as well as infective agents. Therefore, pathogens utilize various methods for acquiring iron from the host ,12. MiniDiagnostic Criteria for Diabetes MellitusDiabetes can be diagnosed by various methods such as hemoglobin A1C (A1C), fasting plasma glucose (FPG), and oral glucose tolerance test (OGTT) .We measure the average blood sugar for the past two to three months using the hemoglobin A1C test. Hemoglobin A1C level in the blood and its interpretation is shown in Table As the name suggests, in the fasting plasma glucose test, we check blood glucose levels after being fasted for at least eight hours before the test. You cannot eat or drink anything but water for at least eight hours beforehand . FastingIn the oral glucose tolerance test, we check blood glucose fasting after two hours of drinking a 75 g of glucose drink. It tells you about how well your body process glucose. OGTT is a more sensitive test than A1C and FPG . The oraMucormycosisA fungal infection caused by a group of molds called Mucoromycetes is known as mucormycosis and is a rare but serious fungal infection. Spores of mucormycosis are present almost everywhere in nature, such as soil and decaying organic matter . The intMucormycosis can manifest in various ways in humans, depending on the location it infects. It can present as pulmonary, cutaneous, disseminated, gastrointestinal, and rhino-orbital-cerebral mucormycosis ,17.Rhino-orbital-cerebral mucormycosis is an infection that involves sinuses, orbits, and the brain. It usually starts by infecting sinuses and finally affects the eyes and the brain if not intervene promptly. It is most common in patients with uncontrolled DM and kidney transplant patients . A most In pulmonary mucormycosis, lungs are infected by inhalation of spores. Cancer and organ transplant patients are at increased risk for pulmonary mucormycosis. Patients suffering from pulmonary mucormycosis have pneumonia-like systems such as shortness of breath, fever, and chest pain . On imagCutaneous mucormycosis is an infection that involves the skin. When the integrity of the skin is damaged, it allows the fungus to enter inside. Skin integrity is altered in a burn patient. Surgery, cut, and other skin traumas also harm skin integrity. In cutaneous mucormycosis, the patient had blisters or ulcers along with pain, redness, and swelling around it .When Mucorales enter the blood circulation and spread to the various body parts, it is termed disseminated mucormycosis. If it extends to the brain, patients have altered mental status and coma. Patients with significant immunosuppression and profound iron overload are at risk of developing a disseminated infection .Gastrointestinal mucormycosis presents as abdominal pain and GI bleeding. Its risk is increased in infants who are premature and have low birth weight if they had surgery, medication, and antibiotics since it weakens the immune system. It can also present in adults if they have diabetes mellitus or undergoing peritoneal dialysis .Management of MucormycosisEarly diagnosis and rapid management through various methods such as antifungal therapy, surgical intervention, and the use of hyperbaric oxygen play a crucial role in the management of mucormycosis. Rapid intervention helps in reducing morbidity and mortality associated with mucormycosis significantly ,20.Surgical debridement of necrotic or infected tissue greatly reduces mortality ,22. PareCombined and aggressive use of surgical debridement, antifungal therapy, and hyperbaric oxygen can significantly and drastically improve the outcome in patients suffering from mucormycosis ,27.The findings of the studies included in this review regarding the association of mucormycosis in diabetes patients are summarized below in Table LimitationsThis study has its limitation due to the minimal number of articles published exclusively illustrating the direct relationship between mucormycosis and DM.The impact of diabetes mellitus on the quality of life of millions of individuals is significant. Susceptibility or increased risk of infection in DM is due to poor immune response and readily available free iron in circulation, which is much more aggravated by high plasma glucose levels and acidic pH. Severe consequences of recurrent as well as rare infections are highly noticeable. One such rare infection which can result in disastrous consequences is mucormycosis. Factors such as decreased number of T lymphocytes, neutrophilic dysfunction, leukocytes apoptosis, and impaired dendritic cell function are the culprits behind severe complications associated with mucormycosis in DM. Earlier research shows that DM patients are more susceptible to severe complications related to mucormycosis than healthy individuals. Thus, there is a need to increase awareness regarding mucormycosis infection and its impact on health in the community to reduce the burden of disease.