Mucormycosis is an invasive fungal infection with high morbidity and mortality rate despite the early diagnosis and proper therapeutic interventions. Given the importance of epidemiological data in reviewing the attitude toward infectious diseases in developing countries, the current retrospective case study aimed to compare the epidemiological aspects, risk factors, clinical characteristics, therapeutic interventions, and outcomes of mucormycosis between adults and children during eight years (2013–2021) in the main infectious disease referral centers in the southwest of Iran. The median age of 164 patients included in this study was 47 years (IQR 22–59). The median length of hospitalization was 33 days.The annual incidence of mucormycosis-related hospitalizations was estimated 1.76 per 10,000 admissions during the study period. Moreover, the incidence of infection was 2.4 times higher in males than females in children. Diabetes mellitus was the most frequent predisposing factor in adults (46.0%). The main risk factor in children was hematologic malignancy (52.6%), but a considerable proportion of them (28.9%) were immunocompetent.The most frequent antifungal agent used was liposomal amphotericin B (82.3%) as monotherapy. The combination therapy was used more in adults (15.8%) than children (7.9%). In addition, surgical intervention with antifungal therapy was considered the most effective therapeutic approach. The in-hospital mortality rate was 14.6% for adults, whereas it was zero for children. Our findings provide a recent epidemiologic analysis of mucormycosis among hospitalized patients in both children and adults. Mucormycosis mainly affects individuals with diabetes mellitus or hematological malignancies and presents as rhino-orbito-cerebral form. Proven diagnosis of mucormycosis according to clinical manifestations and histopathology observations accompanied by proper antifungal treatments may improve survival rates. Mucormycosis affects all age groups, and is associated with significant morbidity and death in both immunocompromised and immunocompetent hosts, depending on the underlying risk factors, site of infection, and early management2.Fungal infections by Mucoralean fungi, namely mucormycosis, are serious and progressive diseases that get increasingly worse3.Studies from all over the world indicate uncontrolled diabetes and hematologic malignancies are predominant risk factors for mucormycosis. The other common predisposing conditions associated with mucormycosis include solid organ/hematopoietic stem cell transplantation, neutropenia, a prolonged period of steroid therapy, treatment with iron chelators such asdeferoxamine, extensive burns, and traumatic events5.Despite the extended dispersion of Mucorales spores in the environment, most species are not capable of infecting humans because they could not proliferate at body temperature. Humans can acquire the infection predominantly through inhalation of spores, occasionally by ingestion of contaminated food or traumatic inoculation. After germination of the spores, sprout branch-like hyphae invade blood vessels and cause necrosis of the surrounding tissues6.The most infected sites in mucormycosis are paranasal sinuses; however, lungs, skin, and brain are other organs that can be infected with mucormycosis. Either the Gastrointestinal system or kidneys are other internal organs that are less likely to become involved7.Standard approaches for the treatment of mucormycosis include a combination of antifungal therapy and surgical intervention to remove infected tissues, especially in rhino-orbito-cerebral form, and control the underlying condition. Liposomal amphotericin B (LAMB) is the first choice that is preferable to the conventional form due to less toxicity and side effects. Moreover, such triazoles as posaconazole or isavuconazole have shown desirable antifungal activity against mucormycosis8. Although, the prevalence of mucormycosis is estimated 50 times higher than global data in developing countries, such as Mexico and India10. Recently, there has been an increase in the incidence of the infection which is linked to increased immunocompromised patients and the presence of various underlying conditions3. Although the number ofcase reports inchildren is growing11, there is not acomprehensive study comparing the epidemiological pattern of mucormycosisin adults and children population. In this regard, the aim of this retrospective case study is the comparison of epidemiological aspects, risk factors, clinical characteristics, diagnosis, therapeutic interventions, and outcomes of mucormycosis between adults and children for eight years (2013–2021) in the main infectious disease referral hospitals in the southwest of Iran as the one of an endemic zone in the Middle East.The epidemiology of mucormycosis has been investigated in previous studies in different world regions, so it has been reported that the prevalence of mucormycosis is about 0.16 cases per 10,000 discharges among the American populationIn total, 164 patients with confirmed mucormycosis were included in this study among 927,404 total hospitalized patients during the study period. The number of patients in children and adults population was 38 (23.2%) and 126 (76.8%) cases, respectively.Of patients, 91 (55.5%) were male and 73 (44.5%) were female . Median age of all patients included in this study was 47 years (IQR 22–59). The mean age of adult patients (> 18 years old) was 51.83 ± 15.21, and the mean age of patients in children group (< 18 years old) was 8.10 ± 5.40 years. The median length of stay in hospital was 33 days.In children, the incidence of infection was 2.4 times higher in males than in females, while this ratio was almost the same in the adult population. The adult to children mucormycosis ratio was 3.3:1.According to our results, the annual incidence  of mucormycosis-related hospitalizations was estimated to be 1.76 per 10,000 admissions during the study period. Moreover, the annual incidence of mucormycosis varied from 2.78 per 10,000 admissionsin 2016 to 1.04 per 10,000 admissions in 2020. During the study period, an increase in the incidence of mucormycosis was observed in autumn . Demographics and characteristics of patients in the total population and also, based on the age group classification (adults/children) are provided in Table During this study, the most common site of mucormycosis was rhino-orbito-cerebral involvement which manifest in 158 (96.4%) patients. Excep than one gastrointestinal mucormycosis reported in one adult patient, rhino-orbito-cerebral involvement was the only clinical form that was obsereved in other patients with no underlying conditions .Pulmonary mucormycosis were reported in 2 patients (1.2%) with diabetes mellitus: one child with tracheal involvement, and another case in an adult with obstruction of the right main bronchus and small lesions of the carina in the left main bronchus. In one case of gastrointestinal mucormycosis in an adult patient, infection was confined to the terminal ileum, cecum, appendix, and right colon. Although rare clinical forms of mucormycosis were not observed in children, they were reported in 3 adult patients in this study as follows: one case of iatrogenic mucormycosis resulted in fungus ball formation in the brain following surgery,  one case of abscess formation in the neck and lower jaw, and one caseof abscess formation in the pterygoid muscle. Also,the most common form of infection in children population of this study was rhino-orbito-cerebral mucormycosis .In the total population of our study, the main predisposing factor for mucormycosis were hematological malignancies  and diabetes mellitus .Statistical analysis of data showed a significant relationship between predisposing factors and the age grouping of patients . Diabetes mellitus  and hematological malignancies  were the most frequent predisposing factors in adults. Hematological malignancies  were the main risk factor in children, althougha high proportion of children  in our study were immunocompetent or no risk factor was found among them.Of those patients with hematological malignancy, AML was diagnosed in 35 cases (21.3%) following with ALL , CLL , with non-Hodgkin Lymphoma .Chronic corticosteroid use , iatrogenic trauma  following tooth extraction or maxillofacialsurgery, and solid organ transplantation  were other risk factors. Moreover, chronic kidney disease (CKD), thalassemia major, and long-term use of Deferoxamine were reported as underlying conditions in 7 (4.3%) patients.Pain, swelling, and protrusion of the eye  were the most common symptoms among the rhino-orbito-cerebral forms of mucormycosis at the time of diagnosis, followed by dirty or bloody nasal discharge , with or without obstruction in the nasal cavities. Significant reduction in the vision of the affected eye was considered a complication of mucormycosis in 3 patients with rhino-orbito-cerebral form. Other symptoms of rhino-orbito-cerebral mucormycosis included headache , swelling and facial pain , decreased level of consciousness , fever , nasal cavity and nasal septum ulcer , and hard palate ulcer , respectively. Among those who presented facial pain and swelling, two patients had facial nerve palsy, and one had abducens nerve palsy. Cough, shortness of breath, and hemoptysis were common symptoms of pulmonary mucormycosis. In the only case of gastrointestinal mucormycosis reported in this study, abdominal pain was the most important symptom of the infection.Radiological evidence was used as an initial diagnostic evaluation in patients who suspected mucormycosis. According to radiological data, thickening of sinusoidal mucosa was observed in 96.3% of the rhino-orbito-cerebral forms of mucormycosis cases. Moreover, complete obstruction of sinuses, bone erosion or destruction, presence of retro-orbital mass, and invasion of nerves or ocular tissue was reported as common clinical features of mucormycosis are associated with sinusoidal and rhino-orbito-cerebral involvements. After the thickening of sinusoidal mucosa, the presence of retro-orbital mass was one of the radiological observations that were significant in the children population . Radiological assessment of other forms of mucormycosis cases declared the presence of mass or abscess in the infected tissue. Also, inflammation of the mucosal membrane or the presence of nodules was reported in flexible bronchoscopy in gastrointestinal involvement.Laboratory-based diagnosis of infection including histopathological examination on biopsy of the necrotic mass or mucosal membrane was performed with H&E and/or PAS staining. In this regard, interstitial inflammation with non-septate fungal hyphae was remarked in reported results.The therapeutic approaches for the treatment of our cases included sinus and ocular decompression, mass resection of the affected sites, control of underlying factors including diabetes and neutropenia, and intravenous antifungal therapy. Table The most frequently antifungal agent used was Liposomal Amphotericin B (LAMB) as monotherapy in 141 (86%) patients of the total population. Table Survival to hospital discharge was considered the primary outcome. According to the results, the rate of survival to hospital discharge in our study was 82.9%. The in-hospital mortality rate in the total population was 14.6%. Moreover, the mortality rate in adults was 19%, whereas it was zero in children .Regarding clinical signs, adult patients with a decreased level of consciousness showed a poor prognosis and a significant correlation with mortality .Patients with rhino-orbito-cerebral mucormycosis had the highest mortality rate , while no mortality was reported among other clinical forms of mucormycosis such as pulmonary, gastrointestinal, and rare clinical forms of mucormycosis.12. To the best of our knowledge, this research is one of the largest studies that provides a comparison of epidemiology and clinical aspects of mucormycosis between children and adults in a developing country such as Iran.Mucormycosis is an invasive fungal infection in immunocompromised patients with high mortality despite early diagnosis and timely therapeutic intervention. During the two last decades, the studies have shown an increase in mucormycosis compared to other invasive mycoses such as aspergillosis among people withimmune deficiencies13. According to the previous study by Kontoyiannis et al., which was conducted in the United States, the incidence of mucormycosis-related hospitalizations was estimated to be 0.12 per 10,000 discharges from January 2005 to June 201414. However, a recent study has declared an 80 times higher prevalence of mucormycosis in India than those developed countries15. From the results of this study, the annual incidence of mucormycosis was 1.76 per 10,000 hospitalized cases, which is a little more than that reported by Hedayati et al. from Iran in 201816.Our results support the data previously reported by Vaezi et al. that has presented Fars province (Shiraz) as the second province in Iran in terms of the high prevalence of mucormycosis17.Although mucormycosis has a worldwide occurrence, its frequency is variable in different world regions.The exact incidence of mucormycosis is not clear, and this was partly because there are a few population-based studiesthat differ in capture periods, populations, and definition or diagnostic procedures18. In the current study, children consist of approximately one-fourth (23.2%) of the total populationwith mucormycosis which was more than in the previous study11.Regarding demographic characteristics of our patients who suffered mucormycosis, we showed the occurrence of the disease in a wide spectrum of ages from childhood to old age. According to previous reports, mucormycosis is a rare infection in children; hence, there are few case studies in the literature17, while the reported mean age varies from 50 to 60 years in other studies in Iran21. A male predominance was noted during the study similar to most of the other previous studies21; however, the sex difference was significant among children. Some authors have correlated this tendency with the protective effect of estrogens, but this correlation may not be valid in children younger than 12 years old11.In agreement with the investigation by Vaezi et al. average age of cases in this study was almost 40 years23. As declared previously, environmental factors such as temperature and humidity are affected the mold distribution in the air resulting in seasonal variation of exposure to spores24.The prevalence of mucormycosis in our study is higher in autumn in accordance with other investigations28. In accordance with previous investigations, the current study showed diabetes mellitus is the most relevant predisposing factor for mucormycosis in adults.Based on studies in different parts of Iran and other countries in the world, diabetes mellitus enumerates as the most common underlying conditions for mucormycosis32. Considering our results, hematologic malignancies and diabetes mellitus are identified almost in equal amounts as the most important underlying conditions in the development of mucormycosis. Since neutrophils have played a crucial role in the innate immune function that is responsible for engulfing and killing pathogenic microbes and invading the germinated form of Mucorales spores, reduced chemotactic activity in diabetic patients and prolonged neutropenia in hematologic malignancies favor the formation of hyphal structures and resulted in the development of mucormycosis in these vulnerable groups33. Overall, any disorders or organ failure could emerge as a potential factor for mucormycosis. In this regard, we found that impairment in the human immune system in patients receiving corticosteroid and solid organ transplant recipients or iatrogenic inoculation could be considered a predisposing factor for mucormycosis similar to other investigations17.Furthermore, investigations admitted differences in the epidemiology of mucormycosis between developed and developing countries which declared patients with hematological malignancies are at greater risk for mucormycosis in Europe and the United States17. One major finding in our study is that a significant proportion of mucormycosis has been reported in immunocompetent hosts  in agreement with other previous studies30. Additionally, Pana et al. have reported invasive mucormycosis in 9.5% of their study population with no underlying medical condition34. These findings are comparable to those of our study, and the difference in the results may be due to the difference in the patient population. In total, mucormycosis in immunocompetent individuals has a worldwide distribution regarding the analysis of literature reviews. Furtheremore, our results support the data previously reported that cutaneous mucormycosis and rhino-orbito-cerebral form are the most prevalent forms among patients with no underlying conditions35.Based on our findings, rhino-orbito-cerebral form was the most common form of mucormycosis in both population that are in accordance with previous literatures34.In the children population, our results are in agreement with a previous study by Pana et al. who have declared that hematologic malignancies are a significant risk factor for mucormycosis in children36 and Nidhi et al.37 that reported orbital and gastrointestinal mucormycosis in immunocompetent children, respectively.It is noteworthy that almost a third of our children population  is immunocompetent hosts. These results support the data previously reported by Amanati et al.36. Evidence explains that mucormycosis mostly can be acquired by inhalation of sporangiospores, wherase ingestion or direct inoculation of spores can lead to gastrointestinal or cutaneous infections. Insect bites, stings, and even bird pecking have all been linked to cases of cutaneous mucormycosis in the past38. Therefore, determining the precise case histories in future investigations may reveal new aspects of epidemiology of mucormycosis.The way of getting infection or transmission of infection in immunocompetent individuals is not clearly known. In this regards, a recent study have reported cases of mucormycosis in immunocompetent children with previous exposure to plants and fodder in infected childrenOther clinical forms as gastrointestinal  was observed in immunocompetent patients in this study. Therefore, possible host and pathogen factors or mode of transmission the spores which can cause infection in individuals without predisposing conditions should be investigated in future studies.39. Regarding the distribution of spores in soil and decayed materials, inhaled sporangiospores can deposit in the nose or paranasalsinuses resulting in rhino-orbito-cerebral mucormycosis.The host immune system and portal of fungi entry may be responsible for the type of infection presentation. Similar to other studies, rhino-orbito-cerebral involvement was the most common form of infection. Also, in accordance with other previous studies, rhino-orbito-cerebral involvement is the most common clinical manifestation in hematologic malignancy and diabetic patients31, no case of cutaneous involvement was reported in our study. In this regards, it should be declared that separate hospitals provide pertinent services for trauma and burnning that were not included in this study.Cutaneous mucormycosis arise as a result of inoculation of spores in the skin layers or blood dissemination from infected organs. Although blood dissemination to skin tissue is rare, spore inoculation can result in cutaneous forms after trauma, burn, or iatrogenic injury. Although cutaneous manifestation has been reported as one of the common clinical forms of mucormycosis in previous studies,especially in immunocompetent individuals40. Furthermore, the presence of the nodule, mass, halo sign, ground-glass opacity, and the air crescent sign is radiologic evidence of respiratory/gastrointestinal tract fungal infections which should be remembered in the diagnosis of pulmonary/gastrointestinal mucormycosis41.In this study, clinical features were one of the most important pieces of evidence which contributed to the diagnosis of infection in all cases. According to our data, most patients with rhino-orbito-cerebralmucormycosis showed clinical signs such as facial pain, swelling, neurologic disorders, nasal ulcer, etc. Hence, vision and facial nerve disorders, the presence of a necrotic scar in maxillary, facial, or sino-orbital tissues in patients with uncontrolled diabetes, immunocompromised status, and, hematological malignancy could alert the physicians in order to early diagnose mucormycosis42.It should be noted that facial nerve palsy, an uncommon clinical manifestation in patients with rhino-orbito-cerebral mucormycosis regarding previous reports, has been announced in 2 patients in our study population43. At the same time, deformation of fungal hyphae due to various factors such as pressures exerted on the fungi by the tissue or folding during processing could result in artificial lines confused with septations and misidentification of fungal agents41. On the other hand,species identification and antifungal susceptibility profiles of fungal agents are valuable aspects in infectious diseases to identify the possible resistant species and geographical distribution of causative agents which is possible using culture of clinical samples.No information about the culture of patients' clinical samples was recorded in the medical record. According to a previous study, culture is performed to diagnose about 69% of cases, worldwide6. Since 2% of patients were diagnosed based on the culture method in Iran according to the previous investigation17, it is concluded that the culture method is not widely used to diagnose mucormycosis in Iran which requires more consideration.Histopathological examination of clinical specimens is the only laboratory method used in the diagnosis of all patients in this study that confirms the mucormycosis. It is worth noting that the histopathology method has still considerable value in the diagnosis of fungal infections. Observation of the non-septate or ribbon-like hyphae in tissue using histopathological examination classifies the infection as proven according to European Organisation for Research and Treatment of Cancer/ Mycoses Study Group (EORTC/MSG) criteria for mucormycosis infection41. Also, posaconazole is recommended for patients with drug intolerance and salvage therapy to prevent recurrence or combination therapy45.Treatment guidelines documented by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) recommend surgical resection in addition to immediate antifungal therapy with liposomal or lipid-complex amphotericin B for adults and children with mucormycosis et al. also declared that initial monotherapy with liposomal amphotericin B is an effective approach to control mucormycosis and decrease mortality46. Given the low mortality rate of patients in this study, appropriate antifungal treatment accompanied by surgical intervention lead to improved outcomes in agreement with previous studies49. Indeed, drug penetration into the damaged tissue enhances by removing the necrotic tissue by surgical excision which causes the effective function of treatment. However, some previous studies have shown that surgery does not significantly affect decreased mortality in patients with mucormycosis50.Most of the patients in the present study received LAMB (86%) considering the first-choice antifungal for management of mucormycosis. Evidence by Jeong51. However, previous research shows that combination therapy did not result in less mortality in comparison with LAMB monotherapy46.At the same time, the combination of LAMB plus azoles or caspofungin has been prescribed for a few patients. Antifungal therapy with LAMB plus caspofungin represents a promising successful treatment for rhino-orbito-cerebral mucormycosis in a study done by Reed et al.46. However, it is not clear which form of amphotericin B has been used in this study; lipid formulation is more favorable due to less toxicity and side effects52.According to a study done by Jeong et al., there is no difference in survival between lipid-based formulation and deoxycholate formulation of amphotericin B53.In our study, the average dose of LAMB administered in children was 5–10 mg/kg/day. Due to the lack of defined optimal dosage of LAMB in children,the average dosage is often extrapolated from adult trials. In this regard, the recommended dosage by studies is 3–15 mg/kg/day54. As a consequence of the unavailability of posaconazole in our country during recent years which leads to low levels of consumption in our study, it was not possible to compare the efficacy of posaconazole for the management of mucormycosis in adults and children. Moreover, isavuconazole is another therapeutic agent that is not available in our country's pharmaceutical market.Posaconazole is another broad-spectrum triazole with significant antifungal activity against Mucorales fungi56. Low in-hospital mortality rate has been reported in some earlier studies58.According to our results, the in-hospital mortality rate was 14.6%. However, the overall mortality rate of mucormycosis has been reported to range from 21.4 to 46.7% in previous studies58.Therefore, if patients could have been followed up for a longer period of time, the mortality rate might have increased. (2) Lower mortality rates have been observed in cutaneous infections; therefore, the variable mortality rates reported in different studies may be related to the site of infection57. (3) Underlying conditions plays a crucial role in mortality rate of mucormycosis. As previously stated, immunocompetent status, or absence of trauma were related to the highest survival rates41. Therefore, a low mortality rate has been observed in this study might be a result of the small number of HSCT patients and CNS mucormycosis among our research population. (4) In addition, it is possible that some of the cases of mucormycosis that leads to death, might not have been properly diagnosed.Low mortality rate compared to other studies may be due to several reasons: (1) The reported mortality rate in our study is based on the in-hospital mortality, and excludes mortality data following discharge. As previously declared by Multani and their collegeues, 1-year mortality rate increased significantly compared to in-hospital mortality59. In our investigation, no case of premature neonates or disseminated mucormycosis were found in the children's group. Besides, lack of reported in-hospital mortality among children may be linked to high proportion of immunocompetent conditions of studied population.In our study, the children mortality rate was zero. In addition to those mentioned above, prematurity of the neonate, age less than 1 year, and the development of the disseminated form of mucormycosis have also been declared as risk factors for mortality in several studies conducted in the population ˂ 18 yearsVarious major limitations must be acknowledged in this study. In the current study, mortality information is confined to hospital lenght of stay. Moreover, diagnosis of mucormycosis was performed based on clinical features and histological examination. As a result, there was no specific culture methods for species identification and antifungal susceptibility testing of isolates. Hence, there is no available data about species distribution or resistance patterns of causative agents. Another limitation is the unavailability of such drug formulation in our country as isavuconazole,making it practically impossible to evaluate the efficacy ofthis treatment.Although further explorations are needed to evaluate the efficacy of new therapeutic agents against fungal infections, it could be claimed that amphotericin B has been an effective antifungal for the management of mucormycosis, so far. With the hope of increasing awareness of fungal infections, the culture of clinical specimens, antifungal susceptibility testing, and molecular-based methods should give more attention as a particular subject in the diagnosis and management of fungal infections.As far as we know, the current study is the first survey pointing to the epidemiological aspects, clinical presentation, therapeutic interventions, and outcomes of mucormycosis with a comparison between adults and children in Southwest Iran during the 8 years (2013–2021). A change in the epidemiology of mucormycosis has occurred in recent years due to increasing at-risk populations, especially in developing countries. In this study, diabetes mellitus and hematological malignancies were found as the most predisposing condition. Also, the rhino-orbito-cerebral clinical type was the most frequent form in patients. Early diagnosis and prompt clinical intervention may resulted in increased survival rate.After being approved by the Ethics Committee of Shiraz University of Medical Sciences (IR. SUMS.MED.REC.1399.321), this multicenter retrospective study was performed from March 2013 through April 2021 in four main referral centers for infectious diseases in Shiraz, Iran  which affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. These hospitals provide specialty and subspecialty services such as surgical, internal medicine, hematology/oncology, and ophthalmology/ENT for children and adults. Namazi and Shahid Faghihi are subspecialty hospitals as the largest referral medical centers in the south of Iran with 824 and 479 beds, respectively, and the average annual admission is more than 50,000/year. Also, Amir and Khalili hospitals as a specialized center for hematology/oncology and ophthalmology/ENT patients with 108 and 86 beds, respectively. In addition, all methods were performed in accordance with the relevant guidelines and regulations, and informed consent was obtained from all subjects and/or their legal guardians.43. All registered data with diagnosis of mucormycosis, zygomycosis, or Mucorales infection were reviewed. Also, the data were reevaluated by infectious disease specialist in order to prevent data missing. The patients with a length of hospital stay fewer than 48 h because of not eligibility to follow up the clinical outcomes or patients with unavailable/incomplete data were excluded from the study. In data collection process, patients with admission in different centers were considered as one patient according to identical national code.As defined by the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria, all children and adults admitted and managed in studied centers with proven mucormycosis based on histopathology reports were includedThen, all demographic information including sex, age, season/year of infection, underlying condition, and comorbid diseases as well as clinical data such as type of mucormycosis (site of infection), clinical presentation, radiological findings, laboratory-based diagnostic findings, surgical interventions, type, dose and duration of antifungal treatment, and clinical outcomes were extracted from the electronic database of hospitals by two physicians.The annual incidence of mucormycosis was calculated annually by dividing the number of cases by the total number of hospitalizations observed in all centers in the same year.Collected data were analyzed by SPSS software (IBM Corp. Released 2013. IBM SPSS Statistics for Windows). Measurement data were expressed as mean ± standard deviation, as appropriate. For categorical variables in 2 × 2 tables, if there was a cell with expected frequencies of less than five, Fisher’s exact test was used. However, if there was not such a cell, the Chi-squared test with continuity correction was used. Comparisons were performed using Student’s t-test for continuous variables. It should be mentioned that a p-value of less than 0.05 was considered statistically significant.


Trends in Hearing. doi:10.1177/2331216518783608Timmer, B.H.B., Hickson, L., & Launer, S. (January 2018). Do Hearing Aids AddressReal-World Hearing Difficulties for Adults With Mild Hearing Impairment? Results From a PilotStudy Using Ecological Momentary Assessment. In the above article Michael David is to be included as third author with the affiliation asLouise Hickson – School of Health and Rehabilitation Sciences, The University of Queensland,Australia.Hence, the author list will be,Barbra H. B. Timmer, Louise Hickson, Michael David, Stefan Launer


The aim of this letter to the editor is to discuss the etiology and pathophysiology of chronic inflammatory demyelinating polyneuropathy (CIDP) in two patients, of whom one experienced a mildly symptomatic SARS-CoV-2 infection 2 months prior to onset of the CIDP (patient-1), whereas patient-2 developed CIDP with bilateral facial palsy 19 days after receiving a shot of an RNA-based anti-SARS-CoV-2 vaccine. Causality between the SARS-CoV-2 infection and CIDP in patient-1 remains unsupported and the diagnosis CIDP in patient-2 remains questionable. Although delineation between CIDP and GBS is not clear cut, bilateral facial palsy and absence of proximal involvement suggest GBS rather than CIDP. With interest we read the article by Fotiadou and colleagues about two patients with chronic inflammatory demyelinating polyneuropathy (CIDP) after an infection with SARS-CoV-2 2 months earlier patient-1) and 19 days after vaccination with an mRNA-based anti-SARS-CoV-2 vaccine (Ad26.COV2.S) [ and 19 dWe disagree with the diagnosis CIDP in patient-2 . The patWe also disagree with the description in Table 2 that involvement of respiratory muscles is absent in CIDP . AlthougWe should be informed if dysarthria was due to facial diplegia, involvement of cranial nerves IX and X, or if dysarthria was due to involvement of the brainstem. A subtype of GBS manifests with bulbar involvement  . MissingFurthermore, we disagree with the notion that CIDP in patient-1 was due to an infection with SARS-CoV-2 [Missing in patient-1 is the determination of cytokines, chemokines, and glial fibrillary acidic protein (GFAP) in patient-1. These CSF parameters have been reported elevated in GBS due to an infection with SARS-CoV-2 .Missing are also the results of contactin and neurofascin antibody titers to assess if the two index cases had a nodopathy rather than a SARS-CoV-2 infection/vaccination-related disease.Overall, the elegant study has several limitations that challenge the results and their interpretation. These limitations should be addressed to broaden the conclusions. Delineation between CIDP and GBS is challenging. Involvement of cranial nerves rather suggests GBS than CIDP.


Inonotus hispidus is a valuable and rare edible and medicinal mushroom with extremely high nutritional and medicinal value. However, there is no holistic insight to elucidate the molecular basis of the differentiated usage and accurate annotation of physiological maturity to fluctuating yields and quality. This study aimed to figure out the fruiting bodies' metabolites change regulation and potential maturating indicators to distinguish different quality I. hispidus. We applied non-targeted ultra-high performance liquid chromatography and high-resolution mass spectrometry combined and with multivariate analysis and analyzed cultivated and wild mushroom I. hispidus in different growth periods . With the fruiting bodies maturating, 1358 metabolites were annotated, 822 and 833 metabolites abundances changed greater than or equal to 1 time from the budding period to the aging period in abundance in cultivated and wild, the total polysaccharides, crude fat, total flavonoids, and total terpenes increased at first and then decreased. Total amino acids, crude protein, and total polyphenols decreased, while the total steroids increased linearly. The change of metabolites showed certain regularity. Metabolic pathways enrichment analysis showed that these metabolites are involved in glycolysis, biosynthesis of amino acids, organic acid metabolism, glycine-serine-and-threonine metabolism, tricarboxylic acid cycle, purine metabolism, and pyrimidine metabolism. In addition, ergosterol peroxide and (22E)-ergosta-4,6,8(14),22-tetraen-3-one can be used as indicator compounds, and their contents increase linearly with the fruiting bodies of I. hispidus’ physiological maturation. This comprehensive analysis will help to evaluate the edible values and facilitate exploitation in mushroom I. hispidus. Inonotus hispidus (Bull.: Fr.) P. Karst. (I. hispidus) is an edible and medicinal mushroom, and the fruiting body is not only used as a food material but commonly soaked in water as a health drink in China1. It is a traditional Chinese medicine "Sanghuang" that was described in ancient Chinese Materia medica books in China, such as Shennong's Classic of Materia Medica and Compendium of Materia Medica. It is used in folk culture as an astringent, a diuretic, and for the treatment of mouth ulcers and inflammation2. Novel scientific investigations revealed anti-tumor4, improving human immunity5, anti-inflammatory6, antioxidant7, antiviral8, and hypolipidemic activity9, and it has great potential application value and development potential in the field of functional food and medicine. The appearance of the fruiting bodies of I. hispidus is golden in the budding period, which can be used as delicious food, but the yield is low. The fruiting bodies in the mature period appears yellowish-brown, while the fruiting body in the aging period contains a large amount of melanin10, which is grayish-black, and the yield is relatively high, but the harvest time is longer than the budding period. As to the fruiting bodies of I. hispidus in different growth periods, the pharmacological effects are also different.The mushroom I. hispidus, there is a lack of research on the variation of metabolite abundance in different growth periods of I. hispidus. Because of the lack of measurable indicators that can characterize the physiological maturity of I. hispidus, which leads to the subjective evaluation of the physiological maturity and the fluctuation of the yield and quality of I. hispidus and the difference in production efficiency of different producers. The quality of I. hispidus can be understood in this way, the fruiting bodies with various metabolic components that can meet the industrial needs of pharmaceutical production are considered to be high-quality I. hispidus samples. For example, in industrial production, the main purpose is to obtain the total amino acid components in the fruiting bodies of I. hispidus, then the fruiting body with a relatively high content of total amino acids is considered to be of high quality. The high quality of I. hispidus fruiting bodies can’t be effectively selected in functional food, medical research and production practice, which has become one of the important factors restricting industrial production. Therefore, there is an urgent need for an indicator of fruiting bodies' maturation, to provide a basis for production and research.At present, in the research and commercial production of functional food related to 13. It aims to detect the overall trajectory of endogenous metabolites in organisms or cells under specific conditions to reflect the pathological and physiological processes of organisms, and some differential metabolites detected have become potential markers that characterize the pathological and physiological states of organisms14. Metabolomics has been widely used in many fields, such as animal and plant metabolism16, microbial metabolism18, disease diagnosis21, and drug development22. In recent years, metabolomics technology has been gradually applied to the field of edible and medicinal mushrooms to study metabolic profiling25. Those studies were carried out scientifically by the method of metabolomics, such as analyzing the metabolic profiling changes in mycelia collected at Pleurotus tuoliensis day 0, and day 35 under different temperatures (17 and 29 °C) by using GC–MS-based metabolomics26, and targeted metabolic profiling annotate changes in the lenticular edodes mycelial metabolome under high-temperature stress27.Metabolomics is a new discipline that has developed rapidly following genomics, transcriptomics, and proteomicsI. hispidus mushroom has largely remained unexplored, and the grading has been confused by producers, traders, consumers, researchers, and related regulatory bodies. Almost all of them in the market were objective for discrimination of growth period based on phenotypic information and physical properties such as individual weight, tissue size, color, water content, etc. We hypothesized that the different qualities of I. hispidus differ in their metabolomic profiles, and they could be distinguished by their chemical constituents.So far, the metabolic profiling of different periods of I. hispidus. Through the potential indicator compounds of physiological maturity of it as a reference, to determine the best harvest time and guide the production practice, to maximize the yield and quality. We found that two metabolites were closely related to their physiological maturity, indicating that they have the potential to be used as markers to distinguish different quality grades. The result was helpful to evaluate I. hispidus fruiting body quality and facilitate exploitation in its consumption and processing.This study aimed to figure out the fruiting bodies' metabolites change regulation and potential maturating indicators to distinguish different quality I. hispidus in the budding period (WB), wild I. hispidus in the mature period (WM), artificial cultivated I. hispidus in the budding period (AB), and cultivated I. hispidus in the mature period (AM) are clustered together closely, while the samples of wild I. hispidus in aging period (WA) and cultivated I. hispidus in aging period (AA) deviate slightly. These results are consistent with our previous conclusion that there are great differences in the types of metabolites in different growth periods of the fruiting bodies of I. hispidus, and there are great differences in the abundance of metabolites between the fruiting bodies in the aging period and the other two periods. The wild and cultivated I. hispidus mushroom in different growth periods as shown in Fig. First, unsupervised Principal Component Analysis (PCA) was applied to assess the overall distribution among all samples and the stability of the entire analysis process. As shown in Fig. B, all thI. hispidus, and the metabolic differences among 6 groups of samples, the supervised partial least square discriminant analysis model was used to further optimize the population separation of fruiting bodies. The comparison between paired AB, AM, and AA samples of Partial Least-Squares Discriminant Analysis (PLS-DA) fruiting bodies showed that there were significant differences in metabolism among different categories in each pairwise comparison of the first component , and 24 oligopeptides were annotated in mushroom I. hispidus fruiting body, most steroids and phenols metabolites increased over the whole period. Most flavonoid metabolites increased at first and then decreased. Among sterols and their derivatives metabolites, the abundance of ergosta-5,7,9(11),22-tetraen-3-beta-ol increased at first and then decreased in cultivated I. hispidus. But in the wild I. hispidus increased with the fruiting body physiological maturity. However, the abundance of ergosterol peroxide of cultivated I. hispidus decreased in the whole period. The abundance of ouabain of wild I. hispidus increased at first and then decreased, but in wild samples, it decreased first and then increased. These changes will directly affect the quality of I. hispidus in different growth periods. Detailed changes about sterols, phenols, flavonoids, and terpenoids metabolites in mushroom I. hispidus are shown in Table With the physiological maturity of the I. hispidus fruiting body, the metabolites showed a regular change trend, and this changing trend occurred not only in cultivated I. hispidus, but also in wild. The results showed that the fruiting body of I. hispidus is in the process from the budding period to the mature period and then to the aging period, the abundance of total polysaccharides, crude fat, total flavonoids, and total terpenes increased at first and then decreased. Interestingly, total amino acids, crude protein, and total polyphenols decreased during the growth periods, while the total steroids increased linearly. The analysis of the content and change regular of the metabolites showed distinct changes in abundance in cultivated and wild. From an overall point of view, the changes of metabolites in cultivated were more stable and the content of metabolites higher than the wild ones. Therefore, we speculate that the cultivated I. hispidus may be more suitable for the function food or medicines. The 8 kinds of primary and secondary metabolites changed trend were shown in Fig. With the physiological maturity of the I. hispidus in the bud stage, mature stage, and aging stage  according to the method reported in the literature. During the period of day 1 to day 20, the fruiting body of end Fig. A.Figure I. hispidus mushroom. The compound (22E)-ergosta-4,6,8(14),22-tetraen-3-one has strong fluorescence under 365 nm ultraviolet lamp, so it was easy to be identified and determined. The content was 0.001% at the budding period on day 10 of the cultivated fruiting body, 0.002% on day 30 of maturity, and only 0.003% on day 50 aging period. In this study, the contents of ergosterol peroxide, and (22E)-ergosta-4,6,8(14),22-tetraen-3-one in the fruiting bodies of wild and cultivated I. hispidus showed some regular and linear changes in the process of physiological maturation -ergosta-4,6,8(14),22-tetraen-3-one gradually increased with the physiological maturity of I. hispidus. The results showed that there were significant differences in the composition of metabolites in the fruiting bodies of I. hispidus in different growth periods, which would lead to different medicinal values. Therefore, different growth periods have a significant impact on the quality of it, and it is necessary to further study and clarify the differences. In our analysis, a total of 1358 metabolites were annotated from I. hispidus fruiting bodies. Based on the metabolic profile analysis of the KEGG database (https://www.genome.jp/kegg/pathway.html) and LIPIDMaps database (http://www.lipidmaps.org/), including 20 sugars, 12 amino acids, 24 oligopeptides, 23 steroids, 31 phenols, 12 flavones, 4 isoflavones, and 10 flavonoids and 11 terpenes were annotated. There are significant differences in metabolites in the process of physiological maturation of mushroom I. hispidus, which is mainly reflected in the change of abundance rather than in kinds.This work investigated the fruiting bodies' metabolites change regulation and potential maturating indicators to distinguish different quality I. hispidus, sugars, amino acids, oligopeptides, steroids, polyphenols, flavonoids, and terpenoids have been reported to have pharmacology activity. Therefore, these metabolites are mainly listed in this paper. Sugars are one of the effective components in I. hispidus, modern pharmacological studies have shown that I. hispidus sugars have protective effects on acute alcoholic liver injury in mice1. Amino acids as one of the important indicators of food nutritional value evaluation29, and have many pharmacological activities, such as L-leucine is used in the clinical treatment of liver disease30. Modern pharmacological studies show that serine plays a role in relieving chronic low-back and knee pain in adults31, leucine can by promoting insulin and Glucagon-Like Peptide 1 (GLP-1) secretion. With physiological maturity, the relative abundance of amino acids such as lysine, threonine, asparagine, serine and glutamic acid and histidine in the fruiting body decreased gradually. The oligopeptide is a biochemical substance between amino acid and protein, which is composed of 2 to 15 amino acids. In this study, it is found that the content of oligopeptides is abundant, especially with the physiological maturity of the fruiting body, the abundance of Glu-Val-Phe increases significantly. On the contrary, the abundance of Tyr-Tyr decreases significantly with the physiological maturity of the fruiting body. Steroids and phenols in I. hispidus are important active components, which have antioxidants2, anti-inflammatory6, and bacteriostatic activities6. In recent years, flavonoids and terpenoids as two important kinds of metabolites in I. hispidus have been widely concerned and studied2. Modern pharmacological studies have shown that flavonoids have antioxidant potential and antimicrobial activity32. Some terpenoids have the effects of antioxidation, liver protection, immunity enhancement, anti-tumor, and so on. Such as betulin, which has anti-inflammatory33, antiviral34, and other activities35. With physiological maturity, the relative abundance of betulin in the fruiting body decreased gradually. These findings suggest that the abundance of metabolites showed some regular changes with the fruiting bodies maturating of I. hispidus. When using these fruiting bodies as raw materials for functional foods and drugs, special attention should be paid to the changes of the abundance of metabolites in the fruiting bodies of I. hispidus.Among the metabolites of I. hispidus mushroom during different growth stages were compared and analyzed. The result showed that the abundance of total polysaccharides, crude fat, total flavonoids, and total terpenes increased at first and then decreased. Interestingly, total amino acids, crude protein, and total polyphenols decreased during the growth periods, while the total steroids increased linearly. In this study, it is worth noting that the content of 8 kinds of primary and secondary metabolites of cultivated I. hispidus mushroom is significantly higher than that of wild, which may be related to the fact that water, temperature, nutrients, and other factors in the artificial cultivation environment are more suitable for the growth of it. Compared with the growth environment conditions of wild I. hispidus, cultivated has more stable growth environment conditions, and is more stable and unified in soil nutritional status, temperature, humidity, precipitation, and so on. These may be some important reasons for the high quality of cultivated. Metabolite profiles are highly affected by environmental conditions, but it needs to be explained by further research. On the one hand, the changes of metabolites in cultivated were more stable and the content of metabolites higher than the wild ones, which is more suitable for the function of food and medicine. On the other hand, cultivated I. hispidus is easier to control the growth environment conditions, standardized production, and easy to obtain high yield. These results suggested that cultivated I. hispidus may be more suitable for the needs of industrial products. From the perspective of primary metabolites and secondary metabolites, the relative contents of total amino acids and total polysaccharides decrease gradually with the physiological maturity of fruiting bodies, if the sugars or amino acids in I. hispidus are used in industry, then the maximum benefit may be obtained in the bud stage, of course, the yield needs to be considered. If steroids are used in industry, and the content of steroids increases with the physiological maturity of the I. hispidus fruiting bodies, then the aging I. hispidus fruiting bodies may be more suitable. This study showed that there were significant differences in the relative abundance of primary and secondary metabolites in the fruiting bodies of I. hispidus in different growth periods. This study provides a scientific basis for selecting the best I. hispidus fruiting bodies in the growing period according to the demand.From the perspective of primary metabolites and secondary metabolites, the changes of eight main metabolites of wild and cultivated I. hispidus, leads to the subjective evaluation of the physiological maturity and the fluctuation of the yield and quality of I. hispidus and the difference of production efficiency of different producers. Therefore, there is an urgent need for an indicator of fruiting body maturation, to provide a basis for production and research. In our study, the content of ergosterol peroxide and (22E)-ergosta-4,6,8(14),22-tetraen-3-one in fruiting bodies showed a regular change during physiological maturation. Almost no ergosterol peroxide and (22E)-ergosta-4,6,8(14),22-tetraen-3-one were detected in fruiting bodies collected on day 0 of the wild and cultivated I. hispidus, but its content was extremely high in physiologically mature fruiting bodies. We deduced that ergosterol peroxide and (22E)-ergosta-4,6,8(14),22-tetraen-3-one may be used as potential indicators to identify whether fruiting bodies have reached physiological maturity. However, the utility of ergosterol peroxide and (22E)-ergosta-4,6,8(14),22-tetraen-3-one content as an indicator of the physiological maturation of mycelia was not determined, and this question merits additional fruiting experiments.In addition, a lack of measurable indicators that can characterize the physiological maturity of I. hispidus mushroom has the same 1358 metabolites, but the metabolites showed distinct changes in abundance in cultivated and wild. It is worth noting that mass spectrometry can’t detect all the metabolites, not because the mass spectrometry is not sensitive enough, but because mass spectrometry can only detect ionized substances, but some metabolites can’t be ionized in the mass spectrometer. Nuclear magnetic resonance (NMR) is needed to make up for the lack of chromatography in the future, and further research is needed38. Through the metabonomic and natural product research combined, the content of ergosterol peroxide and (22E)-ergosta-4,6,8(14),22-tetraen-3-one showed a linear trend increase, which indicated the feasibility of their use as an indicator for fruiting body maturation, but whether this indicator can be applied for the commercial production of mushroom I. hispidus requires the verification of large-scale fruiting experiences. The metabolites of I. hispidus fruiting bodies were different in different growth stages, so the quality was different. However, the maturity of the fruiting body can be determined by peroxidation of ergosterol and (22E)-ergosta-4,6,8(14),22-tetraen-3-one, and the quality of the fruiting body can be screened indirectly. Our research provided an atheoretical basis for quality evaluation and comprehensive utilization of the mushroom I. hispidus from cultivated and wild.In summary, this study showed that with the fruiting bodies maturating, the abundance of metabolites showed some regular changes. The cultivated and wild All chemicals used in this study were of chromatographic grade for liquid chromatography. Acetonitrile, methanol, and formic acid were purchased from Merck . Acetic acid and methyl alcohol were from Tedia . Deionized water was purified by a Milli-Q water purification system .I. hispidus mushrooms were collected from the ancient mulberry protection forest in Xiajin County, Dezhou City, Shandong Province, China . Samples were randomly collected and immediately frozen in liquid nitrogen. The cultivated I. hispidus mushroom was collected from Xiajin Sanghuang Biotechnology Co., Ltd. 6 replicate samples (sampling position: middle part) were collected at each time point and frozen in liquid nitrogen immediately. All samples are stored at − 80 °C until metabolomics analysis. The specimens (No. HMJAU58767) are deposited in the Key Laboratory of Medicinal Fungal Resources and Development and Utilization, Jilin agricultural university.Wild g, 4 °C for 20 min. To reduce the matrix effect, better detect metabolites and optimize the peak shape, 300 μL supernatant and 150 μL LC–MS grade water. were diluted to 53% methanol content. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15,000×g, 4 °C for 20 min. Finally, the supernatant was injected into the UHPLC-MS/MS system analysis39.Crushed tissues (100 mg) were placed in the Eppendorf tube (EP tube) and resuspended with prechilled 80% methanol and 0.1% formic acid. After the vortex shock, the samples were incubated on ice for 5 min and then were centrifuged at 15,000×40. UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system  coupled with an Orbitrap Q Exactive HF mass spectrometer . Samples were injected onto a Hypesil Gold column  using a 17 min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for the negative polarity mode were eluent A  and eluent B (Methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min; 100–2% B, 14.1 min; 2% B, 17 min. Q Exactive HF mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, capillary temperature of 320 °C, sheath gas flow rate of 40 arb, and aux gas flow rate of 10 arb.UHPLC-MS/MS analysis refers to previously methodsI. hispidus in different growth periods, the complex chemical composition and its change trend line were shown, but the practical application may involve a macroscopic overall quantitative analysis. Therefore, the contents of total polysaccharides, total amino acids, crude protein, total steroids, polyphenols, total flavonoids, terpenoids, and crude fat in wild and cultivated I. hispidus fruiting bodies were analyzed in this study.Through the analysis of the metabolic profiling of the fruiting bodies of wild and cultivated 23. The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.1  to perform peak alignment, peak picking, and quantitation for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, 100,000. After that, peak intensities were normalized to the total spectral intensity. According to the formal definition of metabolites annotation and annotation developed by the Chemical Analysis Working Group of the Metabolomics Standards Initiative (MSI) working Group on Chemical Analysis41, this study annotates metabolites detected by non-targeted metabonomic scans. The normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. And then peaks were matched with the mzCloud (https://www.mzcloud.org/), mzVault, and MassList database to obtain the accurate qualitative and relative quantitative results. Statistical analyses were performed using the statistical software R (R version R-3.4.3), Python (Python 2.7.6 version), and CentOS (CentOS release 6.6), When data were not normally distributed, normal transformations were attempted using of area normalization method.Data processing and metabolite annotation refer to previously methods44. These metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/pathway.html), Human Metabolome Database (HMDB) (https://hmdb.ca/metabolites), and LIPIDMaps database (http://www.lipidmaps.org/).Data were normalized for internal consistency by processing the constant weight of the solvent per volume of each sample. The internal consistency of the data is normalized data were scaled to the median value for each compound, then missing values were imputed with the minimum detected value for that compound. Moreover, a variety of curation procedures were carried out to ensure that a high-quality data set was made available for statistical analysis and data interpretation. The QC and curation processes were designed to ensure accurate and consistent annotation of true chemical entities and to remove those representing system artifacts, misassignments, and background noise. Library matches for each compound were checked for each sample and corrected if necessary. Metabonomic data analysis refers to previously methodsThe data analysis of PCA and PLS-DA were performed using software python (Python-3.5.0), and the charts were drawn by software R (R-3.4.3). We applied univariate analysis (t-test) to calculate the statistical significance . The metabolites with VIP > 1 and P-value < 0.05 and fold change ≥ 2 or FC ≤ 0.5 were considered to be differential metabolites. Volcano plots were used to filter metabolites of interest-based on log2(FoldChange) and -log10 of metabolites.For clustering heat maps, the data were normalized using z-scores of the intensity areas of differential metabolites and were plotted by the Pheatmap package in R language. A statistically significant correlation between differential metabolites was calculated by R language. P-value < 0.05 was considered as statistically significant and correlation plots were plotted by corrplot package in R language. The metabolic pathways enrichment of differential metabolites was performed, when the ratio was satisfied by x/n > y/N, metabolic pathways were considered as enrichment, when the P-value of metabolic pathway < 0.05, metabolic pathways were considered as statistically significant enrichment. For line charts and tables, the data were normalized using Microsoft Office Excel 2019 software, and the chemical structure formula was made by ChemDraw 15 software.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


This study aimed to assess the correlation between residual hearing and audiologic outcomes after cochlear implant (CI) surgery in patients with cochlear nerve deficiency (CND).This retrospective study included 57 patients with CND who underwent CI surgery. Patients were divided into four groups according to hearing level  and three groups according to residual hearing  based on the measured response at each frequency. Auditory performance  and speech perception  were assessed before and 2 years after the surgery.Forty‐seven (82.5%) patients had complete or total hearing loss (≥95 dB) and 17 (29.8%) had no spectrum hearing before CI surgery. Twenty‐nine (50.9%) patients did not exhibit residual hearing at 4 kHz. All patients demonstrated an improvement in auditory performance and speech perception: the CAP score in the 80–95 dB group was significantly higher than that in the 110–120 and >120 dB groups, and the entire spectrum hearing group showed significantly higher CAP, SIR, and IT‐MAIS scores than the partial spectrum hearing group and significantly higher CAP, SIR, IT‐MAIS, and MUSS scores than the no spectrum hearing group.For patients with CND, residual hearing, especially high‐frequency residual hearing, was poor and postoperative audiologic outcomes were significantly associated with the range of residual hearing.4 We thus analyzed the correlation between residual hearing and audiologic outcomes in a relatively large study sample.22.1We retrospectively analyzed the data of 57 patients with CND and profound‐to‐complete SNHL who underwent CI surgery at Tongren Hospital in Beijing between January 2015 and December 2019. All patients underwent unilateral CI surgery. The ear with better residual hearing with hearing aids (HAs) was selected for CI surgery. The use of human subjects in this study was approved by the Research Ethics Board of Tongren Hospital, Beijing, China.The inclusion criteria were as follows: (1) severe‐to‐complete SNHL, (2) preoperative direct oblique sagittal 3D MRI scans perpendicular to the long axis showing either a thin CN or no CN at the fundus of the internal auditory canal (IAC), (3) no syndromes, (4) a minimum of 3 months of experience wearing HAs and language training before CI surgery (CI was indicated if the patients responded to sound), and (5) successful CI surgery and activation at 1 month postoperation.2.2The diagnostic protocol for children with suspected hearing loss involves audiological tests, including measurements of the behavioral observation audiometry (BOA), acoustic emittance, distortion product optoacoustic otoacoustic emission, auditory brainstem response, cochlear microphonics, and 40‐Hz auditory‐evoked related potential. BOA is used to observe changes in auditory behavior in response to stimuli in infants and young children aged <6 months. Visual reinforcement audiometry trains children to establish conditioned reflexes and turn to a light box when hearing the stimulus sound and is suitable for children aged 7 months to 2.5 years. Play audiometry involves simple games during which children respond clearly upon hearing sounds.Hearing levels were recorded at 0.5, 1, 2, and 4 kHz using BOA. A lack of measurable response was considered indicative of no residual hearing. To calculate the average hearing level, a lack of measurable response was assumed to be 5 dB‐HL greater than the maximum output of the audiometer (125 dB). The average hearing threshold was averaged across 0.5, 1.0, 2.0, and 4.0 kHz on BOA. Hearing impairment was classified in accordance with the World Report on Hearing by the World Health Organization in 2021 as follows: mild (20–35 dB), moderate (35–50 dB), moderately severe (50–65 dB), severe (65–80 dB), profound (80–95 dB), and complete or total hearing loss (≥95 dB).Due to a high percentage of complete or total hearing loss in our cohort, patients were divided into four groups according to the hearing level as follows: 80–95, 95–110, 110–120, and >120 dB. Based on the measured response at each frequency, patients were divided into three groups according to the residual hearing as follows: entire spectrum hearing, partial spectrum hearing, and no spectrum hearing. Entire spectrum hearing indicated that patients responded at all frequencies during the tests; partial spectrum hearing indicated that patients did not respond to at least one frequency during the tests, and no spectrum hearing indicated that patients did not respond to any of the frequencies during the tests.2.3High‐resolution computed tomography (HRCT) was performed using a 64‐slice computed tomography (CT) scanner . Volumetric acquisitions were contiguously reconstructed using a 1 mm‐slice thickness throughout the temporal bone. HRCT was used to evaluate IEM according to Sennaroglu's classification.2.4Auditory performance before and 2 years after CI surgery was evaluated using categorical auditory performance (CAP), speech intelligibility rating (SIR), the Infant–Toddler Meaningful Auditory Integration Scale (IT‐MAIS) for patients aged <3 years, and the meaningful use of speech scale (MUSS).CAP is an index with eight levels of sound perception (0–7) ranging from “no awareness of the environment” (0) to the “use of telephone with known users” (7). CAP is intended to reflect the real‐life auditory capabilities of children. The SIR is a highly reliable and efficient measure of children's speech production in real‐life situations and ranks a child's spontaneous speech into five categories ranging from “connected speech is unintelligible” (1) to “connected speech is intelligible to all listeners” (5). The IT‐MAIS with scores ranging from 0 to 40 is used to assess auditory performance including changes in vocalization, spontaneous alerting to sounds, and the ability to derive meaning from sounds. The MUSS is a parental reporting scale comprising 10 questions that is used to determine the frequency of use of speech in children's day‐to‐day behavior.2.5t‐test. An analysis of variance was performed to determine the differences among the residual hearing groups. All statistical tests were two‐tailed, and p < .05 was considered significant.SPSS statistics software  was used for data analysis. Descriptive statistics were used to determine the medians, ranges, means, and standard errors of the mean (SEMs). The difference between pre‐ and post‐CI surgery audiologic performance was determined using a paired‐samples 33.1Fifty‐seven patients  with CND who underwent CI surgery were included in the study. All patients had congenital SNHL and had failed newborn hearing screening. Custom electrodes were used for patients with modiolar deficiency‐type IEM  and lateral wall electrodes were used for patients with normal cochlea. The mean age (±SEM) at implantation was 32.72 ± 2.17 months . All patients underwent unilateral CI surgery. The ear with better residual hearing with HAs was selected for CI surgery. The left and right ears were implanted in 39 and 18 patients, respectively. Thirty patients were implanted with MED‐EL  devices, 16 with Cochlear  devices, nine with AB  devices, and two with Nurotron  devices. Sixteen patients (28.1%) had IEM. Fifteen patients (26.3%) had a narrow IAC. Besides the patients with common cavity and incomplete partition‐III (IP‐III), 45  of the patients had a narrow BCNC. Twenty‐one (36.8%) patients were fitted with CIs bimodally. The patients' demographic information is shown in Tables 3.2The median average hearing threshold of the patients was 111.25 dB  as shown in Figure The hearing levels at frequencies of 0.5, 1, 2, and 4 kHz are shown in Figure Among the 57 patients, 28 (49.1%), 12 (21.1%), and 17 (29.8%) were included in the entire spectrum, partial spectrum, and no spectrum hearing groups, respectively Figure . In the 3.3p < .001). All hearing level subgroups showed significant improvements (p < .05), as shown in Figure p < .01 and p < .05, respectively). All hearing spectrum subgroups . Compared to the entire spectrum hearing group, the partial spectrum hearing and no spectrum hearing groups showed significantly poorer postoperative CAP scores .The CAP and IT‐MAIS were used to assess auditory performance before and after CI surgery. Overall, the CAP score before and after CI surgery was 0.54 ± 0.09 and 4.26 ± 1.25, respectively. The difference was significant, as shown in Figure s Figure  showed sp < .001) from 1.63 ± 0.35 to 26.60 ± 1.34  .All patients showed significant improvements in IT‐MAIS scores  Figure . The ent3.4p < .01) .We used the SIR and MUSS scores to evaluate speech performance. All patients had a preoperative SIR score of 1. The postoperative SIR score was 1.98 ± 0.11. The SIR scores for the overall cohort and hearing level and hearing spectrum subgroups were significantly different before and after CI surgery  Figure . Howeverp < .01)  Figure . Among t) Figure .4The present study demonstrated that the condition of residual hearing was significantly associated with postoperative audiologic outcomes after CI surgery.Patients with CND display limited residual hearing. In a previous study, >70% of ears with CND showed profound hearing loss.Due to the limitations of imaging techniques that are currently available, the CN may not be directly visible on MRI. The IAC and BCNC indirectly determine the condition of the CN. IAC stenosis is often considered to be associated with CND.Children with residual hearing have a higher likelihood of early auditory development than those with no measurable thresholds, leading to better auditory performance after CI surgery.The range of residual hearing could be used to better predict postoperative CI outcomes. A previous study demonstrated differences in the performance of children with 70 dB‐HL of residual hearing (up to 1 kHz) and those with only low‐frequency residual hearing (up to 0.5 kHz) and reported that children with a wider frequency range of hearing showed better performance.In cases where the assessment process reveals no evidence that a CI would provide any benefit or would provide inadequate benefits, an auditory brainstem implant (ABI) should be considered. CND is considered an indication for ABI surgery.5This is the first study to assess the correlation between residual hearing and auditory performance after CI surgery in patients with CND. In addition to the average hearing threshold, close attention should be paid to the range of residual hearing. Residual hearing, especially high‐frequency residual hearing, is poor in patients with CND, and the CI outcomes in patients with CND are variable. The current study showed that postoperative audiologic outcomes in patients with CND were significantly associated with the range of residual hearing. Furthermore, the evaluation of residual hearing using behavioral audiometry may provide useful information that can facilitate preoperative counseling for these patients.Simeng Lu analyzed the patients' cochlear nerve deficiency data and was a major contributor to the writing of the manuscript. Xingmei Wei substantively revised the manuscript. All authors read and approved the final manuscript.None of the authors received any other funding and none have financial relationships or conflicts of interest to disclose.This study was approved by the Research Ethics Board of Tongren Hospital, Beijing, China.The parents of the patients were fully informed about the evaluation prior to participation. All the parents provided written informed consent and participated in the evaluation free from coercion. The patients' information was protected. Written informed consent for the publication of details regarding the patients' clinical status was obtained from the parents.


These women completed self-report telephone questionnaires regarding their hearing difficulties. Some of these women underwent subjective and objective hearing tests. Out of 159 (50.3%) symptomatic women with SBIs, 80 reported hearing impairments, including hearing loss  and tinnitus . Five out of seven (71.4%) women who underwent an audiologic evaluation exhibited hearing loss. Of women who underwent silicone implant removal, 27 out of 47 (57.4%) reported the improvement or resolution of their hearing complaints. In conclusion, hearing impairment is a frequent complaint among symptomatic women with SBIs, and tinnitus was found to be the most common complaint. A significant reduction in hearing difficulties was observed following silicone implant removal. Further studies using larger populations are needed to verify the occurrence of hearing impairments in these women.Many women with silicone breast implants (SBIs) report non-specific complaints, including hearing impairments. Hearing impairment appears to be associated with a number of autoimmune conditions. The current study aimed to evaluate the prevalence and severity of hearing impairments among women with SBIs and to explore potential improvements in their hearing capability following implant removal. Symptomatic women with SBIs ( Silicone is a polymeric compound comprising a silicon–oxygen unit with two organic groups attached to it, thus forming rubber-like materials, which have been used for many medical purposes, such as breast implants, joint implants, testicular prostheses, intraocular implants, artificial cardiac valves, cosmetic rhinoplasty, various shunts and catheters .Silicone breast implants (SBIs) are commonly used for cosmetic purposes or in women requiring breast reconstruction in cases of breast malignancy. The cosmetic use of silicone breast implants was first introduced in 1962. This procedure became extremely popular among women all over the world, and to date, millions of women have undergone silicone breast implantation for cosmetic purposes ,8,9,10. In addition, after several reports of breast-implant-associated anaplastic large-cell lymphoma , the US The connection that links SBIs and autoimmunity may be supported by the concept of autoimmune/inflammatory syndrome induced by adjuvants (Shoenfeld’s ASIA syndrome), which includes several autoimmune conditions that are induced following exposure to substances with adjuvant activity .In a recent large-scale study  of 24,65Furthermore, a previous study  reportedRecently, we studied a large group of women with SBIs who reported various non-specific systemic symptoms, such as chronic fatigue, sleep and memory impairments, myalgia, arthralgia, dry mouth and eye and many more. These symptoms have been previously reported in women with SBIs ,18. HearSensorineural hearing loss (SNHL) is caused by damage to the structures in the inner ear, the auditory nerve, and/or auditory pathways in the central nervous system. SNHL might be genetic or acquired and includes a variety of hearing impairments. Autoimmune inner ear disease is a rare condition that has been defined as progressive, fluctuating unilateral or bilateral SNHL. Its pathogenesis has not been elucidated ,20. ThisTinnitus, the perception of sound in the absence of an external auditory stimulus, may be related to many factors. The sound sensation may be ongoing or pulsatile in its pattern. Tinnitus can be classified as objective or subjective, given the patient’s condition. Tinnitus will be classified as objective if the sound is generated from the patient’s own body and is audible to an examiner. However, subjective tinnitus is much more common, but in this case, there is no inner sound produced by the body but a subjective self-reported sensation .In addition, patients with systemic autoimmune diseases may experience tinnitus as well as vestibular symptoms and muffled hearing . AutoimmTinnitus is a common disorder among adults worldwide, with an estimated prevalence of 10–15% in the US  and withIn addition, although it is a common disorder, it has been shown in the past that there is a link between tinnitus and impaired quality of life as well as symptoms of depression, anxiety and sleep disorders ,40. ThisIn the present study, we aimed to examine whether there is an increase in the incidence of subjective complaints of hearing impairment, in addition to objective tests in SBI women. Moreover, we assessed whether an improvement in hearing impairment was noted following silicone implant removal.Participating women were from all over the State of Israel and complained of non-specific symptoms that appeared in association with silicone breast implants over time. In recent years, the awareness of autoimmune symptoms associated with women with silicone breast implants has increased, and accordingly, women suffering from these clinical manifestations have contacted the Zabludowicz Center for Autoimmune Diseases at the Sheba Medical Center. The inclusion criterion was symptomatic women with a history of breast augmentation surgery (for cosmetic or reconstructive purposes). The exclusion criteria included the removal of SBIs before the initial appointment with the physician at our center.The study population included 160 symptomatic women with silicone breast implants who came to our institute during the years 2019–2021. These women underwent a comprehensive anamnestic questionnaire indicating the diverse systemic symptoms they suffered from, such as fatigue, muscle pain, memory impairment and sleep disturbances, as well as hearing loss, muffled hearing and tinnitus. Among the 160 women, there were women who underwent surgery for cosmetic reasons and for reasons of reconstruction following resection of breast cancer. Their age range was 22–75 with an average age of 43.7 (±10.48).The 160 SBI women who were studied underwent a comprehensive anamnestic questionnaire that included demographic details, previous illnesses and therapy, and family history, as well as the current illness, including the symptoms and time frame of their appearance. The non-specific complaints were numerous and mostly fatigue, muscle and joint pain, memory impairment and sleep disturbances. In addition, among the symptoms the women complained of were hearing loss and tinnitus. The questionnaire also included information about the reason for implantation and time since implantation. Women who complained of hearing loss, muffled hearing and tinnitus provided details regarding their hearing impairments by means of a telephone questionnaire. The phone questionnaire was used due to the COVID-19 pandemic and difficulties meeting the SBI women personally.The questionnaire included two topics. First, there were questions about hearing loss or hearing impairment complaints, such as the self-definition of their hearing sensitivity. The SBI women who complained of tinnitus in the initial interview were asked about their tinnitus characteristics, including the level of nuisance and loudness. In women who underwent implant removal, a further interview concerning improvements in their hearing impairments was conducted.A comprehensive hearing evaluation was performed in seven women who complained of hearing impairments and agreed to be tested. The hearing evaluation included the following: audiometry, a word recognition test, tympanometry, acoustic reflex, transient evoked otoacoustic emissions (TEOAE) and auditory brainstem response (ABR) testing.p < 0.05 was considered statistically significant. Statistical analysis was performed using the IBM SPSS Statistics for windows (Version 23.0) software platform.Continuous variables are presented as means. Ordinal variables are presented as medians. Categorical variables are presented as counts (%). All tests used were two-tailed, and We included several conditions as “hearing impairment” in this study, including hearing loss, muffled hearing, tinnitus and others. Moreover, we categorized hearing impairment into ordinal variables (1–4) based on the hearing impairments reported. The Wilcoxon signed-rank test was used to determine the effect of SBI removal by comparing the number of hearing impairments before and after the removal of SBIs.The mean age of the 160 women was 43.7 (±10.48) years. SBIs were implanted for cosmetic reasons in 128/159 women (80.5%) and for reconstruction purposes in 31/159 women (19.5%). The mean time from SBI implantation to symptom onset was 10.6 (±5.9) years. Of 158 (53.8%) women, 85 underwent the removal of their silicone implants.Of the 159 women, 80 reported hearing impairments (50.3%). For one woman, we were unable to obtain this information.Classified by complaints, we found that out of the women who complained of hearing impairments, 30 (38.4%) reported hearing loss, and 29 (37.1%) reported tinnitus. Furthermore, 2 (2.5%) reported muffled hearing, 10 (12.8%) reported hearing loss combined with tinnitus, 2 (2.5%) reported tinnitus combined with muffled hearing, 4 (5.1%) reported a combination of the three complaints , and 1 (1.2%) reported “disturbed speech perception with multiple speakers”. For two women, we received no further information other than “hearing impairment” .As noted above, 85 patients underwent implant removal. Out of the 80 women who reported hearing impairments, 52 underwent implant removal (65%).Of the 44 (61.3%) women who complained of hearing loss, 27 underwent the surgical removal of their implants. Out of the 27 women whose implants had been removed, 9 (33.3%) reported improvement in or recovery from hearing loss, and 16 (59.2%) reported a lack of improvement. For two (7.4%) women, we were unable to obtain this information.Of the 45 (71.1%) women who complained of tinnitus, 32 underwent the surgical removal of their implants. Out of the 32 women who removed their implants, 22 (68.7%) reported improvement in or recovery from tinnitus, and 9 (28.1%) reported a lack of improvement. For one (3.1%) woman, we were unable to obtain this information.Six of the eight (75%) women who complained of muffled hearing underwent the surgical removal of their implants. Out of the six women who removed their implants, three (50%) reported improvement or recovery, and three (50%) reported a lack of improvement.We focused on the group of women who complained of hearing impairments and also underwent implant removal surgery and compared the reports before and after implant removal. We found that before silicone breast implant removal, 16 (32%) reported hearing loss and 20 (40%) reported tinnitus. In addition, one (2%) reported muffled hearing, seven (14%) reported hearing loss combined with tinnitus, one (2%) reported tinnitus combined with muffled hearing, four (8%) reported a combination of the three complaints  and one (2%) had “disturbed speech perception with multiple speakers” .When we checked the women’s reports after they underwent silicone breast implant removal, we found that 24 (51%) reported no symptoms, 11 (23%) reported hearing loss and 4 (8.5%) reported tinnitus. Furthermore, two (4.2%) reported muffled hearing, four (8.5%) reported hearing loss combined with tinnitus, one (2.1%) reported a combination of the three complaints  and one (2.1%) had “disturbed speech perception with multiple speakers” .Among the women who reported hearing impairments and underwent implant removal, 47 (90.3%) of them reported whether a change occurred in their hearing impairments following removal. Out of the 47 women who reported changes, 27 (57.4%) reported improvement in or recovery from their hearing impairments, while 20 (42.6%) reported no change .n = 47; Z = −4.863; p value < 0.0001).Our findings indicate that reported conditions of SBI-related hearing impairments significantly improve after the removal of SBIs  women. It should be noted that tinnitus was one of the most prevalent complaints among the 160 women with SBIs.As for the hearing evaluation , five ouOur retrospective cohort study aimed to evaluate the prevalence of hearing impairments associated with silicone breast implants as part of the ASIA syndrome. Our data showed an increase in self-reported hearing impairments, including hearing loss, muffled hearing and tinnitus among our cohort. Importantly, we found that the removal of SBI resulted in an improvement in hearing impairment. Interestingly, Kim et al.  could noThe results of our study included a few major findings: 50.3% of the woman who arrived at our clinic because they were symptomatic and suffering from clinical manifestations reported hearing abnormalities. Of the women who underwent implant removal surgery, 57.4% reported improvements in or recovery from hearing abnormalities. Focusing on tinnitus, the improvement was greater: 68.7% reported improvement in or recovery from tinnitus after removing their silicone implants.Tinnitus is a slightly confusing symptom, which usually makes it difficult to diagnose. Subjective tinnitus can only be heard by the person suffering from it. Currently, there are no objective diagnostic tools available to make a definite diagnosis of tinnitus. The effects of tinnitus can be assessed through several health questionnaires . TherefoAs shown in Seven women who reported hearing loss underwent a comprehensive audiologic evaluation. Based on audiometry, hearing loss was evident in five of the seven women, most of whom exhibited high-frequency sensory neural hearing loss. This finding is in keeping with the results of a recent review on audiologic manifestations of autoimmune inner ear diseases . IntegraIt is worth mentioning that, as detailed in While providing valuable novel knowledge about hearing abnormalities, including hearing loss and tinnitus, among SBI women, this study has several limitations. One of them is the assessment of hearing abnormality complaints only in a selected group of patients who approached our institute with complaints regarding their medical conditions. This assessment may not reflect the group of SBI women suffering from hearing loss and tinnitus. Moreover, a pre-morbid evaluation was not available; thus, prior hearing loss cannot be ruled out. Finally, a larger group of SBI women should be studied prospectively in order to better characterize hearing impairments.In this study, we found that hearing impairments are commonly reported by symptomatic SBI women. Tinnitus was the most common hearing complaint reported by these women. Using questionnaires and reports collected before and after implant removal surgery, we found that there was a significant improvement in symptoms, with the most notable improvement in self-reported tinnitus. These findings suggest that hearing abnormalities, which include hearing loss, muffled hearing and tinnitus, are causally related to silicone breast implants. The connection between SBI and hearing impairments can be explained by the ASIA syndrome and the “adjuvant hypothesis”, and therefore, the removal of silicone implants may resolve these symptoms. Further studies using larger populations of women with SBIs are needed in order to verify the occurrence of hearing impairments in these women.