Beauveria bassiana is an entomopathogenic fungus used in agriculture as a biological controller worldwide. Despite being a well-studied organism, there are no genomic studies of B. bassiana isolates from Central American and Caribbean countries. This work characterized the functional potential of eight Neotropical isolates and provided an overview of their genomic characteristics, targeting genes associated with pathogenicity, the production of secondary metabolites, and the identification of CAZYmes as tools for future biotechnological applications. In addition, a comparison between these isolates and reference genomes was performed. Differences were observed according to geographical location and the lineages of the B. bassiana complex to which each isolate belonged. Beauveria, about 25 species have been described, most of which originated in Asia—the species’ center of origin [Beauveria bassiana  Vuill  is a versatile microorganism capable of killing insects, surviving as a saprophyte in soil, and creating symbiotic associations with plants as an endophyte [Beauveria bassiana sensu lato has a worldwide distribution and can infect around 700 insect species [Beauveria has been used in the biological control of numerous agricultural pests [Within f origin . Beauverndophyte ,4. Beauv species , Beauveral pests .B. bassiana has a monophyletic origin [Metarhizium anisopliae (Metschn.) Sorokin [According to phylogenetic studies based on the nuclear ribosomal internal transcribed spacer (ITS) and the elongation factor 1-alpha (TEF1-a) gene, c origin . Within c origin . Howeverc origin . The sam Sorokin .B. bassiana from Europe, Asia, Oceania , and North (USA) and South America (Colombia) available in the GenBank database of the National Center for Biotechnology Information ). More recently, eight additional genomes from Central America and the Caribbean, specifically Costa Rica, Honduras and Puerto Rico, have been added to the database [Currently, there are 17 fully sequenced genomes of database , for a tdatabase .B. bassiana for the biological control of different insect pests [Zabrotes subfasciatus (Boh.) [Galleria mellonella (L.); all remaining isolates were extracted from soil in different agricultural systems [B. bassiana fungal accessions from Central America  and the Caribbean (Puerto Rico). Our results provide new insight for the understanding of the pathogenic capacity of the species and the creation of a possible global metagenome. We have identified interesting genetic elements that potentially contribute to the pathogenicity as well as other interesting enzymes that add value to the understanding of the entomopathogenic activity of B. bassiana. Here, we report fungal isolates from the neotropics and provide an overview of the genomic features targeting pathogenic genes and their secondary metabolism potential. We also provide a comparison between these isolates and the previously characterized reference genomes.Costa Rica has implemented entomopathogenic fungi such as ct pests  since thct pests ,14,15. Ts (Boh.) , while fB. bassiana isolates [The fungal isolation, DNA extraction, library prep and sequencing strategy was previously reported for eight isolates . A simplPrior to the de novo genome assembly, raw Illumina reads were quality-checked using FastQC  and procBeauveria bassiana ARSEF 2860 (gb|ADAH00000000.1) reference. To identify the kmer’s distribution through the genomes and to plot its distribution, Jellyfish v.2.2.10 [Genome assemblies were compared and evaluated using Quast v.5.0.2  against v.2.2.10  and Genov.2.2.10  were useB. bassiana genomes: B. bassiana strains ARSEF 2860 (gb|ADAH00000000.1); Bv062 (gb|GCA_003337105.1); B. bassiana JEF-007 (gb|GCA_002871155.1); and B. bassiana HN6 (gb|GCA_014607475.1). To assess the quality and completeness of the assemblies, Quast v.5.0.2 [Due to the short-length nature of Illumina reads and the intrinsic complexity of producing high quality eukaryotic genome assemblies in one go, we implemented the Multi-Draft based Scaffolder (MeDuSa) to optimize the draft assembled genomes scaffolder Medusa v.1.6  against  v.5.0.2  and BUSC v.5.0.2  were useAspergillus oryzae, due to its well-documented necrotrophic lifestyle. In order to identify putative secreted proteins within each fungal isolate, SignalP 4.0 [As a first proteome draft, proteins were predicted by implementing Augustus v2.6.1  trained nalP 4.0  was run B. bassiana isolates [B. bassiana strains ARSEF 2860 (GenBank accession no. ADAH00000000.1); B. bassiana HN6 (GenBank accession no. GCA_014607475.1); and C. militaris CM01 (GenBank ID no. GCA_000225605.1). Single-copy BUSCO genes were identified within each organism and their corresponding proteins were aligned with Multiple Alignment using Fast Fourier Transform (MAFFT) v7.397 [C. militaris CM01 was used [B. bassiana ARSEF 2860 through standard mode parameters.A phylogeny tree was constructed using single-copy BUSCO genes  for all isolates  and refe) v7.397  with the) v7.397 . An autowas used  as the owas used  aided thFor the discovery of the potential biological meaning of the fungal isolates, the BlastKOALA tool was implemented as an automatic annotation server for the genome and metagenome sequences . KEGG orB. bassiana genome annotation for Carbohydrate-Active Enzymes (CAZymes) was performed automatically using the Augustus called-proteins through the dbCAN2 meta server [a server  with thea server  softwareB. bassiana, as there are no entries for entomopathogenic fungi in the PHI-base [B. bassiana isolates and fungal references were identified using homology to characterized MAT genes specific to Clavicipitaceae and Cordyceps sp. using BLASTp. Selected hits fulfill both an e-value of 0.05 or lower and a query coverage filter of at least 55 .To identify the presence of potential virulence-associated genes, blast analysis was conducted against the host–pathogen interactions database (HPIDB 3.0) . The BloPHI-base . Mating-Unprocessed short raw reads were analyzed using Jellyfish and GenomeScope to plot the kmer distribution to check for genome size, heterozygosity rate and repeat content. Globally we observed an optimal genome size, haploidy and low repeat content for all isolates . Aspergillus oryzae dataset, called out 9465 to 10,453 putative proteins. From these, isolates B0, B13, B26 and B27 presented similar counts for potential secreted proteins , strains B1 and B31 had 1028 and 1039, respectively. Similarly, strains B43 and B44 reported 1058 and 1061 ; BbasHN6 (gb|GCA_014607475.1); and C. militaris CM01 (gb|GCA_000225605.1) were included as outputs. This analysis produced a total of 293 core-genes. For each gene, its corresponding BUSCO protein was selected, concatenated and aligned using MAFFT, and an ML tree was inferred using IQ-Tree  were assigned into 10,356 orthogroups. Fifty percent of all genes were assigned in orthogroups with 10 or more genes (G50 was 10) and were contained in the largest 4546 orthogroups (O50 was 4546). There were 7047 orthogroups within all species and 6284 of these consisted entirely of single-copy genes. In addition, there were 1899 unassigned genes accounting for 1.9% of the total number of genes . All isoisolates .To introduce biological information using KEGG categories and the number of gene counts, we focused on specific functional categories . B43 andFrom our analysis, a total of 2349 protein models were assigned to CAZymes distributed into the Auxiliary Activities (AA), Carbohydrate Binding Modules (CBM), Carbohydrate Esterase (CE), Glycoside Hydrolase (GH), Glycosyl Transferase (GT), and Polysaccharide Lyases (PL) families. Most proteins (54%) were associated with the GH family, followed by the GT family (30%) and various other gene families .B. bassiana isolate, we identified 18 putative glycoside hydrolase family 18 (GH18) CAZymes; whereas for the C. militaris CM01 and B. bassiana ARSEF2860 references, we identified 21 genes for each reference. Other important chitinases in the families GH3 and GH16 were absent in our isolates, while for the families GH75 (Chitosan) and GH76, two and nine enzymes were identified, respectively. Furthermore, family GH10 enzymes, present in our isolates, have only been reported in Beauveria bassiana [Cordyceps militaris (L.), Metarhizium anisopliae and Metarhizium acridum (Driver & Milner) J.F. Bisch.), while the GH84 and GH95 families, which were reported previously as exclusive for entomopathogenic fungi [We focused on identifying key members from the GH family, which are involved in the enzymatic deconstruction of chitin  and harbbassiana  and are ic fungi , were obic fungi .C. militaris reported one combination of GH18 + CBM66 , whereas those for Polyketide synthases (PKS) and Terpenes were comparable . All genTo identify the presence of potential virulence-associated genes, a blast analysis was conducted against the Pathogen–Host Interaction database v4.5 (PHIbase). Hits above 70% were the most informative and reliable. We identified PHI genes for important gene families, such as virulence-associated genes, degrading insect cuticles genes, mating-type genes, and core genes for the biosynthesis of secondary metabolites . From thB. bassiana isolate from this study against a database downloaded from the NCBI database specific to the Clavicipitaceae and Cordyceps species. This protein database contained 372 unique entries. We identified seven potential orthologues for each genome respective to the database. Interestingly, each gene was in a single-copy gene orthogroup, except for one orthogroup that lacked a protein for B31, and the genome references B. bassiana ARSEF 2860 and C. militaris CM01; this corresponded to a mating-type protein a-1 (orthogroup OG0009027). On the other hand, the six mating-type candidates had a corresponding single-copy protein in C. militaris, and all homologues showed a high level of conservation  and, at the same time, showed a complete absence of NRPS and PKS compared with the other isolates and reference genomes. In previous analyses, B31 also showed unique alleles in several SSR markers and its lineage could not be determined, while the rest of the isolates were highly diverse but, hence, distinct from Cordyceps [Phylogenetic analyses performed in previous studies determined that all the isolates , except for B27 and B31, belong to an African-Neotropical lineage of bassiana  defined bassiana . In the ordyceps .B. bassiana  and C. militaris (CM01) reference genomes confirmed the singularity of the B31 isolate, whose proteome had a greater similarity to C. militaris than the B. bassiana references and isolates. The direct ancestors of B. bassiana were Asian Cordyceps species, according to Xiao et al. [Beauveria anamorphs and Cordyceps teleomorphs [B. bassiana and the Cordyceps species is the capability of the former to infect a wide host range, while Cordyceps species usually manifest host specificity [Metarhizium species have been shown to evolve from specific-host species to generalist-host species [Beauveria and Cordyceps or a new lineage of the B. bassiana sensu lato complex. New species of Beauveria have been reported in South America [Beauveria species, typically B. bassiana, tends to overshadow the presence of the newly determined species [The Orthofinder ortholog comparison between the fungal isolates and taris CM0 referenceomorphs ,47,48. Ataris CM0 referenc species . This ra America ,51,52 an species ,54.B. bassiana genomes and other entomopathogenic fungi [B. bassiana. In the case of secondary metabolite precursors, the B. bassiana genome contains 45 SM core genes, including 13 NRPS, 12 PKS, 7 NRPS-like, 1 PKS-like, 3 hybrid NRPS-PKS, and 12 genes related to FAS/terpene/steroid biosynthesis [Metarhizium acridum, has been reported by Gao et al. [C. militaris contains enough genes for the infection of specific hosts, while B. bassiana and M. anisopliae are mostly generalist species and have the same NRPSs as the ARSEF2860 reference and fewer PKS components.KEEG analysis of important metabolic pathways, such as the environmental information processing, biosynthesis of secondary metabolites, and lipid and amino acid metabolism, were completed in our genomes. Those pathways are related to the capability of the isolates to develop a pathogenic response and toxin production during the infection process . Previouic fungi , and hero et al. . The reaB. bassiana including one B-type, one C-type and three G-type, and only the C- and G-type showed decreased virulence in topical bioassays [There are no entries for entomopathogenic fungi in the PHI-base , therefoioassays   and other isolates, indicating a possible geographic differentiation. Similarly, continental isolates belonging to different lineages defined by Rehner et al. [Anopheles vectors. Perhaps, due to B31’s association with Costa Rican sugarcane fields, a similar effect is occurring; however, further analysis is needed to investigate this pronounced genetic differentiation.Since allopatric speciation was previously described by Rehner et al.  as an imr et al. , such asr et al. . Accordir et al. , the Bb8Beauveria or other entomopathogenic fungi. Studies from Mexico [Beauveria isolates. Untapped Beauveria diversity in the neotropics through genomics is crucial to develop a comprehensive international landscape of the B. bassiana complex and the different species of the genus. The above, including the finding of Cordyceps ancestor species, is needed to map their evolutionary relationships, host, niche and environmental adaptations.There are still countries, such as Belize, Guatemala, El Salvador, Honduras, Nicaragua, Panama, Dominican Republic, and Haiti, where there are no genomic studies or the molecular characterization of m Mexico ,60, Brazm Mexico  and Costm Mexico ,15,62, h


The purpose of this study was to determine the burden of high frequency noise-induced hearing loss (HFNIHL) in Chinese workers exposed to hazardous noise through meta-analysis, to evaluate the major risk factors of HFNIHL in Chinese workers, and to provide evidence for reducing the risk of HFNIHL. We searched for relevant studies on HFNIHL published between January 1990 and June 2022. Inclusion and exclusion criteria were established to screen the literature, and the quality of the studies was assessed. Meta-analysis was performed using the software Stata 17.0. A total of 39 studies involving 50,526 workers in different industries were included in this study. The incidence of HFNIHL in the noise-exposed group (36.6%) was higher than that in the control group (12.5%), with a pooled odds ratio (OR) of 5.16 and a 95% confidence interval (CI) of 4.10–6.49. Sensitivity analysis showed that the results of this meta-analysis were stable. Funnel plots and Egger’s test showed no publication bias. Subgroup analysis showed heterogeneity among the results of different studies, which was related to gender, publication year, age, length of work, and type of industry. The dose–response analysis showed that cumulative noise exposure (CNE) and length of work were the main risk factors for HFNIHL. This study suggests that the detection rate of HFNIHL is high in Chinese workers, the risk of HFNIHL increases rapidly when CNE reaches 90 dB(A)·year, and the first 15 years of exposure to noise is a period of increasing risk. Therefore, reasonable measures for preventing hearing loss should be taken to reduce the risk of occupational HFNIHL. The burden of disease and death caused by environmental pollution is becoming a public health challenge, especially in developing countries worldwide. Prior to the 1990s, many countries focused on scientific research and policy-making on pollutants such as air pollution, water pollution, and solid waste. In fact, as early as 1971, a working group of the World Health Organization (WHO) concluded that noise was a major threat to human well-being .Hearing loss is defined as a reduced sensitivity to normal sounds. According to the “World Report on Hearing” published by the WHO in 2021, currently more than 1.5 billion people  suffer from hearing loss, and it is estimated that more than 700 million people may have disabled hearing loss by 2050 . HearingCurrently, China is in the stage of accelerating industrialization, with traditional industries and new equipment manufacturing industries expanding and industrial noise increasing, especially the noise generated by machine operations such as air compressors, ventilation fans, textile machines, and metal processing machines. Factors such as a lack of ideal noise reduction designs in factories, lack of protection engineering in workshops, weak awareness of personal noise control and prevention, and prolonged working hours may lead to workers being exposed to hazardous noise environments for a long time. Due to the high incidence of high-frequency hearing loss (HFNIHL) in workers exposed to noise, the purpose of this study is to determine the occupational burden of HFNIHL in Chinese workers exposed to harmful noise levels and to evaluate the main risk factors leading to HFNIHL in Chinese workers, providing a basis for reducing the risk of HFNIHL.The impact of industrial modernization on workers’ hearing loss in China has re-ceived attention. A recent meta-analysis of high-frequency hearing loss in Chinese workers based primarily on the literature from 2017 to 2019 was conducted by Kuang et al. . The resTo objectively understand the burden of occupational noise exposure hearing loss in Chinese workers, this study searched the literature of high-frequency hearing loss studies from January 1990 to June 2022, developed reasonable literature inclusion criteria, conducted a dose–response analysis of risk factors for high-frequency hearing loss, and performed sensitivity tests and publication bias tests on the results of meta-analysis.The search was conducted on both Chinese and English databases, including the China National Knowledge Infrastructure (CNKI), Wanfang Database, Chinese Biomedical Literature Database (CBM), Web of Science, PubMed, and MEDLINE. The search terms “industrial noise”, “occupational noise”, “workers”, “hearing loss”, and “China” were used to search the published literature from January 1990 to June 2022.Inclusion criteria were: (1) studies in both Chinese and English that investigated hearing loss due to occupational noise exposure in China, (2) participants had no other underlying diseases, (3) studies that reported the number of participants exposed to different noise levels and the number of participants with high-frequency noise-induced hearing loss (HFNIHL), and (4) studies that included a control group with a different length of occupational exposure. Exclusion criteria were: (1) studies of noise exposure in workers outside of China, (2) studies of hearing loss or deafness that were not related to noise exposure, (3) studies of non-occupational noise exposure, (4) studies that did not include a noise background control group, (5) animal studies on noise-induced hearing loss (NIHL), (6) studies with incomplete data on the number or prevalence of NIHL cases, (7) studies with evident errors or incomplete data on the demographic characteristics of participants, and (8) books, conference proceedings, and news reports on noise exposure.eq), cumulative noise exposure (CNE), and the prevalence of high-frequency noise-induced hearing loss (HFNIHL).Data extraction included the first author, publication year, sample size, basic demographic information , industry type, equivalent sound levels  , which i2. When I2 ≤ 50%, a fixed-effect model was used for analysis, and when I2 > 50%, a random-effect model was used. Subgroup and sensitivity analyses were performed based on the characteristics of the included studies. Publication bias was assessed using funnel plots and Egger’s regression test.Meta-analysis was conducted using the software Stata 17.0. The effect size was measured using the odds ratio (OR), and a 95% confidence interval (95% CI) was reported. Heterogeneity was assessed using IA total of 704 articles were retrieved from the database search. After screening , 39 studThe basic characteristics of the 39 included articles are shown in 2 = 89.3%, p < 0.001) (p < 0.001).A heterogeneity test showed that there was heterogeneity among the studies (I< 0.001) . Therefo< 0.001)  showed tThe 39 studies included in this meta-analysis were divided into five subgroups based on publication year, gender, mean age, length of employment, and industrial type to explore the contribution of these factors to the heterogeneity among the study results. The results are shown in 2 of 61.8% and p < 0.05. The pooled OR value for studies published after 2010 was 4.29 (95% CI: 3.26–5.64), with an I2 of 90.6% and p < 0.001.Publication year subgroup: The pooled OR value for studies published before 2010 was 7.4 (95% CI: 5.43–10.09), with an I2 of 95% and p < 0.001. The pooled OR value for female workers was 7.89 (95% CI: 3.56–17.48), with an I2 of 68.4% and p < 0.05. The gender composition of the subjects was not specified in 31 studies.Gender subgroup: The pooled OR value for male workers was 12.22 (95% CI: 5.22–28.61), with an I2 of 68.9% and p < 0.001. The pooled OR value for workers over 40 years of age was 5.88 (95% CI: 3.21–10.76), with an I2 of 95.2% and p < 0.001. One study did not specify the age of the subjects.Age subgroup: The pooled OR value for workers under 40 years of age was 4.65 (95% CI: 3.90–5.55), with an I2 of 83.5% and p < 0.001. The pooled OR value for workers with more than 10 years of employment was 5.42 (95% CI: 3.63–8.08), with an I2 of 90.2% and p < 0.001. Three studies did not specify the length of employment of the subjects.Length of employment subgroup: The pooled OR value for workers with less than 10 years of employment was 4.88 (95% CI: 3.63–6.56), with an I2 of 64.7% and p < 0.001. The pooled OR value for the mining industry was 2.29 (95% CI: 0.70–7.58), with an I2 of 95.8% and p < 0.001. Only two studies involved noise exposure in the mining industry, and the heterogeneity was very significant. There was only one study each in the transportation industry and other industries, and they had no statistical significance.Industrial type subgroup: The pooled OR value for the manufacturing industry was 4.62 (95% CI: 3.87–5.51), with an IIt can be seen that heterogeneity in the results of different studies correlated with gender, year of publication, age, duration of employment, and type of industry.The dose–response relationship between cumulative noise exposure (CNE) and length of work with the risk of HFNIHL is a nonlinear curve, which was fitted using a spline function. The dose–response curve between CNE and the risk of HFNIHL is shown in The dose–response curve between length of work and the risk of HFNIHL is shown in Gender was a categorical variable, and age was divided into categorical variables by <30 and ≥30 years. From Due to the inclusion of a large number of studies in this research, to evaluate the stability of the meta-analysis model, as shown in p > 0.05).The funnel plot  construcThis study reviewed and analyzed the literature on high-frequency hearing loss (HFNIHL) caused by occupational noise in China from 1990 to 2022. The meta-analysis results showed that the incidence rate of HFNIHL caused by occupational noise exposure among Chinese workers was 36.6%. Basu et al. conducted a meta-analysis of occupational noise-induced hearing loss in India and found an incidence rate of 50% for NIHL in Indian workers . Mrena eNoise-induced hearing loss (NIHL) initially presents as sensorineural hearing loss in higher frequency ranges (3 kHz–6 kHz), which is known as HFNIHL. As time goes on, hearing loss can gradually develop in lower frequency ranges . In thisNoise-induced hearing loss is irreversible, and there is currently a lack of effective treatments. Therefore, prevention measures are particularly important to reduce the incidence of NIHL. Measures such as strengthening the awareness of noise hazards among enterprise management, setting up necessary noise protection facilities, controlling noise propagation, implementing shift work systems for noise job positions, reducing exposure time in noisy environments, strengthening occupational health education for noise workers, and strictly wearing personal protective equipment are recommended .There was a correlation between noise intensity and high-frequency noise-induced hearing loss (HFNIHL). When studying hearing loss in a boiler factory, Ni et al. found that the detection rates of HFNIHL among workers exposed to noise intensities of 86–90 dB(A) and 101–105 dB(A) were 33% and 78%, respectively . Hu et aThe occupational risk of HFNIHL in workers is related to their length of work, with a higher detection rate of HFNIHL being associated with longer exposure times to noise ,35. LianThe study included different literature with heterogeneity in the results. Pan et al. reported in 2009 that the detection rate of HFNIHL in workers exposed to noise intensity of 85–100 dB(A) was 69.1% . It can Due to the heterogeneity of the studies included in this research, a sensitivity analysis was performed using the one-by-one exclusion method to examine the original meta-analysis results. The results showed that the pooled OR value and 95% CI hardly changed, indicating that the literature screening method, inclusion criteria, and analysis model selection in this study were qualified. Therefore, the conclusions drawn from this research were relatively robust. Funnel plots were constructed, and the Egger linear regression test was used for a qualitative and quantitative evaluation of publication bias, indicating that the conclusions of this study were credible and that publication bias is not present.Hearing protection devices (HPDs) are an effective means of preventing NIHL for workers in the workplace. In 1980, China established a noise standard for industrial workplaces, which states that noise in the workplace must not exceed 85 dB(A). All workers exposed to noise levels above 90 dB(A) are required to wear hearing protection devices (HPDs). Some of the literature in this study reported that HPDs were provided in factories, but only a few workers were willing to wear them ,19. The A systematic review and meta-analysis of Chinese occupational NIHL has been conducted previously by another author ,59. HoweThere are, admittedly, some limitations to this study. All of the included literature are cross-sectional studies, and there is a lack of large sample cohort studies, which may affect the causal relationship judgment of HFNIHL risk. Conference papers, in-house hearing test records for different industrial sectors, and studies in languages other than Chinese and English were not included, which may introduce some bias. In some included studies, the frequency selection for measuring hearing thresholds was not entirely consistent. We hope to conduct some rigorously designed cohort studies of noise exposure and NIHL risk in large samples for different industrial sectors in the future. This will help us further understand the natural patterns of NIHL occurrence and development.The prevalence of HFNIHL in Chinese workers is relatively high (36.6%), and cumulative noise exposure and length of employment are the main risk factors for HFNIHL. In the early stage of noise exposure, the risk of HFNIHL increases slowly, but after reaching the sensitive point of CNE around 95 dB(A)·years, the risk increases rapidly. The first 15 years of noise exposure is a period of increased risk for HFNIHL, after which the risk tends to flatten out.In order to reduce the incidence of occupational HFNIHL, engineering control measures should first be used to reduce noise generation and minimize occupational noise exposure. Secondly, a comprehensive workplace hearing loss prevention plan should be developed, such as noise assessment, noise control, worker hearing monitoring, and appropriate use of hearing protectors. Finally, sound occupational noise control regulations should be established, and regulatory compliance should be checked and monitored by relevant regulatory authorities. This will greatly reduce the burden of occupational noise-induced hearing loss.


We report the case of a 65‐year‐old woman whose colonoscopy revealed a soft submucosal tumor approximately 7 cm in diameter in the ascending colon with an overlying flat lesion. The tumor was diagnosed as a lipoma with an overlying adenoma. Endoscopic submucosal dissection (ESD) was performed. Pathological examination revealed that the epithelium was a low‐grade tubulovillous adenoma, while the submucosal yellow tumor was a lipoma. ESD appears to be a safe and effective treatment for colorectal lipomas overlying lipomas with colorectal adenomas. We report a case of endoscopic submucosal dissection (ESD) of a 7 cm lipoma in the ascending colon with an overlying flat colorectal adenoma. ESD appears to be a safe and effective treatment for an overlying colorectal lipoma with colorectal adenoma. Colorectal lipoma is indicated for treatment of symptomatic cases and cases in which large size and malignancy cannot be ruled out. Surgery has been performed for colorectal lipomas, but in recent years, safer resection by less invasive endoscopic submucosal dissection (ESD) has been reported. Here we report a case in which a colorectal lipoma with an overlying adenoma was resected by ESD, which has not been reported so far.A 65‐year‐old woman presented with a positive fecal occult blood test result. Colonoscopy revealed a soft and yellowish submucosal tumor approximately 7 cm in diameter in the ascending colon, with a flat lesion overlying the tumor Fig. . The flaPathological examination was performed using a 75 × 55 mm specimen. The epithelium was a low‐grade tubulovillous adenoma, while the submucosal yellow tumor was a lipoma. The boundaries between the two were clearly demarcated Fig. .Colorectal lipoma is indicated for treatment of symptomatic cases and cases in which large size and malignancy cannot be ruled out. In this case, the complication caused by the adenoma warranted treatment. Although surgery has been the conventional treatment for such lipomas, recent reportsAlthough ESD of colorectal lipomas has been reported, there are only a few reports of lipomas covering adenoma,Informed consent was obtained from the patient for publication of this case report and accompanying images.


Salacca zalacca) is known for its high antioxidant content and low caffeine levels, making it a promising candidate for the development of value-added health products. However, there is a lack of scientific evidence for its anti-hyperglycemic effects. To address this, we investigated the in vitro and in vivo anti-hyperglycemic and antioxidant effects of salak seed extract. The HPLC chromatogram of salak seed extract shows a prominent peak that corresponds to chlorogenic acid. In vitro studies revealed that salak seeds inhibited α-glucosidase activity and glucose uptake in Caco-2 cells in a concentration-dependent manner, while also exhibiting antioxidant properties. The extract exhibits a non-competitive inhibition on α-glucosidase activity, with an IC50 and Ki of 16.28 ± 7.22 and 24.81 μg/mL, respectively. In vivo studies utilizing streptozotocin-nicotinamide-induced diabetic mice showed that the extract significantly reduced fasting blood glucose (FBG) levels in the oral glucose tolerance test. Continuous administration of the salak seed extract resulted in lower FBG levels by 13.8% as compared with untreated diabetic mice, although this change was not statistically significant. The estimated LD50 value of salak seed extract exceeds 2000 mg/kg, and no toxicity symptoms have been detected. Our research supports that salak seed extract has the potential to serve as a functional food or supplement that may be beneficial in reducing postprandial hyperglycemia among people with type 2 diabetes. This effect was explained by the salak’s inhibitory mechanisms of glucose absorption due to inhibition of both α-glucosidase activity and intestinal glucose uptake, coupled with its antioxidant effects.Salak seed extract ( Salacca zalacca (Gaertn.) Voss) is native to Indonesia, Malaysia, and other Southeast Asian countries, including Thailand, where it is known locally as snake fruit [50) of 16 μg/mL and 20 μg/mL, respectively [Salak , protein (4.22%), and carbohydrates (38.9%), while also containing chlorogenic acid as an active compound . AlthougDespite the efforts to develop functional health products from salak seed waste, a limited number of studies have been conducted on the pharmacological activities of salak seeds, particularly focusing on their antidiabetic potential. The current study aimed to elucidate the antidiabetic activities of salak seed extract and the mechanisms underlying its antihyperglycemic effects, both in vitro and in vivo, utilizing an STZ-nicotinamide (NA)-induced type 2 diabetes mellitus (STZ-NA induced diabetic) mouse model. Furthermore, the current study investigated the antioxidant and lipid peroxidation effects of salak seeds.w/w.The HPLC chromatogram of salak seed extract showed a prominent peak at a retention time of 7.2 min, which corresponds to the chlorogenic acid reference standard . The chl50) values for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays in the salak seed extract, in comparison with those of ascorbic acid used as a positive control. The measured extract exhibited a concentration-dependent inhibitory effect on the generation of malondialdehyde (MDA), as depicted in 50 value of the extract was determined to be 4.34 ± 1.71 µg/mL. In comparison, the positive control, Trolox, revealed an IC50 value of 0.011 ± 0.005 µg/mL.As shown in p < 0.05). The salak seed extract concentration-dependently inhibited glucose uptake in Caco-2 cells compared to the control, with an IC50 of 0.54 ± 0.13 mg/mL.50 values of salak seed extract and acarbose were 16.28 ± 7.22 µg/mL and 370.99 ± 12.43 μg/mL, respectively. max, Km, and Ki, were calculated to be 0.0073 μM/min, 0.82 mM, and 24.81 μg/mL, respectively.Salak seed extract exhibited a concentration-dependent inhibition of α-glucosidase activity, which was higher than that exerted by acarbose (the positive control). The IC50) value of salak seed extract exceeds 2000 mg/kg.Oral administration of salak seed extract at a dose of 2000 mg/kg did not result in any observable symptoms of toxicity or behavioral changes when compared to the control group . Further0–120 min in normal mice treated with salak seed extract at doses of 500 and 1000 mg/kg was comparable to that in the normal control group (ol group C.p < 0.05). A comparison of the AUC0–120 min . In particular, the body weight of the untreated diabetic controls and the groups treated with 500 and 1000 mg/kg of the salak seed extract were significantly decreased in the last week of treatment compared to the baseline (p < 0.05). However, the mice in the normal control and glibenclamide-treated groups exhibited stable, unchanged body weights throughout the experiment. There was a significant increase in the food intake of diabetic mice as compared with that of the normal mice over the treatment period (p < 0.05)  B. Food ip < 0.05) and progressively raised until the end of the study. On the 14th and 28th days of treatment, the administration of 5 mg/kg glibenclamide significantly reduced the average FBG level of STZ-NA-induced diabetic mice with a percentage reduction of 30% and 37.6%, respectively compared to that in the untreated diabetic mice (p < 0.05). After the 14th day of treatment, the doses of 500 and 1000 mg/kg of salak seed extract did not show a significant effect on the FBG of STZ-NA-induced diabetic mice as compared with the untreated diabetic group. However, the FBG levels in DM mice treated with salak seed extract at doses of 500 and 1000 mg/kg for 28 days showed a trend towards being lower than those in the untreated DM mice, approximately 3% and 13%, respectively (As shown in ectively .p < 0.05)  . There wSalak fruit and its seeds are currently processed as waste. However, previous research has shown that the seeds can be used to make salak seed coffee, also known as Salacca coffee, which is a functional beverage ,14,15. TSaleh et al.  reported0–120 min compared with untreated hyperglycemic mice. The anti-hyperglycemic effects of the seed extract were comparable to those of the antidiabetic drug, glibenclamide. Studies have shown that inhibition of α-glucosidase activity improves postprandial hyperglycemia [50 value of 15.94 ± 2.52 μg/mL [50 value of 16.28 ± 7.22 µg/mL. This was more potent than acarbose, suggesting a possible role of salak seed extract as a food supplement for lowering the glycemic index and reducing the risk of type 2 diabetes.This work represents the initial evidence of the hypoglycemic properties of salak seed extract in a murine model of diabetes mellitus caused by streptozotocin-nicotinamide, as assessed using an oral glucose tolerance test, where pretreatment with the extract before glucose loading markedly reduced FBG and AUCglycemia ,18. α-Glglycemia ; however52 μg/mL , salak si value of 24.81 μg/mL. Unlike acarbose, which exhibits competitive inhibition [Using Lineweaver–Burk plots to determine the type of α-glucosidase inhibition, we found that salak seed extract exhibits non-competitive inhibition of the enzyme, with a Khibition , salak sIn addition to the inhibition of α-glucosidase enzyme, the current approach for regulating postprandial blood glucose levels is the suppression of glucose absorption in the small intestine by the inhibition of sodium-dependent glucose transporter 1 (SGLT1) or glucose transporter 2 (GLUT2) . Our stuIt is interesting to note that the salak seed extract did not exhibit a glucose-lowering effect on normoglycemic mice, despite its ability to delay glucose absorption in diabetic mice in the OGTT model. While this study does not provide a definitive explanation for this phenomenon, we speculate that the impact of salak seed extract on glucose metabolism remains within the bounds of normal homeostasis. However, a recent study identified functional and structural changes in type 2 DM mice, including an increase in the number of enterocytes in the villi of the small intestine . This adThe involvement of oxidative stress in the etiology of diabetes suggests that antioxidant therapy has promise for its management. The antioxidant effects of salak seed extract have been verified in previous studies ,21. The Since 500 and 1000 mg/kg doses of the salak seed extract were shown to be effective for its anti-hyperglycemic actions in the present OGTT model, the same doses were subsequently employed in the 28-day continuous oral administration study. In contrast to the postprandial anti-hyperglycemic effects observed in the OGTT model, however, continuous oral administration of the extract did not produce a statistically significant antidiabetic effect during the period tested, albeit with an FBG-lowing tendency . A prior50 value. After 14 days of oral administration of 2000 mg/kg of salak seed extract to 5 female mice, it was determined that the body weight of the mice remained unaffected when compared to the control group. Furthermore, the administration of salak seed extract at the dose of 2000 mg/kg did not result in any mortality during the entire duration of the study, and no significant behavioral changes were observed. These findings suggest that the estimated LD50 value of salak seed extract exceeds 2000 mg/kg, indicating a low level of toxicity.In a previous study , rats weMedicinal plants have been employed in managing type 2 diabetes due to their fewer or lack of side effects when compared to pharmaceuticals. Consumption of tea, coffee, or decaffeinated coffee has been shown to reduce the risk of type 2 DM . This coSalak seeds from the Sala NoenWong cultivar were collected from a fruit garden in the Chanthaburi province of Thailand in May 2021 and authenticated at the Faculty of Pharmaceutical Sciences, Burapha University (sample specimen BV-210501).® filter paper no. 1 , and the residue was extracted again in the same way. The pooled filtrates from each extraction were dried using a vacuum evaporator at 50 °C under reduced pressure, and the resulting extracts were stored in airtight containers at 4 °C. Only analytical-grade chemicals, solvents, and reagents were used in this study.The plant’s seeds were rinsed with tap water, chopped into small pieces, and dried in a hot air oven at 55 °C for 24 h. The dried seeds were then pulverized into a powder using a grinder and passed through a 0.5 mm sieve. Extraction was conducted using a microwave-assisted extraction (MAE) technique with a solid-to-liquid ratio of 1:40. The solvent was 75% ethanol. The MAE  technique utilizes a temperature-controlled system, which is set to reach 120 °C within 3 min and held at 120 °C for 10 min. The resulting sample was filtered through Whatman® C18 column  with a C18 guard column  was utilized. The detailed HPLC conditions used were the same with a previous study [Quantification of chlorogenic acid in salak seed extract was coinducted using a high-performance liquid chromatography (HPLC) method  on a Shius study . A calibThe method of Sato et al.  was usedc and As are the absorbances of the control and the sample, respectively.The antioxidant activity of salak seed extract was evaluated using DPPH, ABTS, and FRAP assays, as previously described . AscorbiThen, the IC50 values of DPPH and ABTS were calculated by plotting the percentage of inhibition against the log concentration of the salak seed extract.4 was employed as the standard. The samples were evaluated in triplicate and the Fe2+ content was given as the mean ± SD of FeSO4 equivalent per gram of extract.For the FRAP assay, the extract’s ferric-reducing capacity was measured using the method described by Vongsak et al. . FeSO4 wc and As are the absorbances of the control and the sample, respectively.Malondialdehyde is a major byproduct of lipid peroxidation during oxidative stress. MDA is often used as a marker of oxidative stress, which has been implicated in the pathophysiology of diabetes and its complications. ,30. The 50 value was examined from the MDA production percentage inhibition activity versus the salak seed extract concentration.The resulting MDA was quantified as MDA equivalent (mg MDA/g linoleic acid). The IC® HTB-37™) for in vitro anti-hyperglycemic evaluation, and subsequently an MTT assay to evaluate the cell viability under the exposure of salak seed extract, according to a previous method [We conducted Caco-2 cell culture (ATCCs method . Brieflys method :(3)Cell The cultured Caco-2 cells were incubated for 2 h with glucose-free Dulbecco’s modified Eagle medium (DMEM) and then for 30 min with Hanks’ balanced salt solution (HBSS) . The celc and As are the absorbances of the control and the sample, respectively.The inhibitory effect of the salak seed extract on α-glucosidase activity was determined using a spectrophotometric method, according to a previous study . The per50 value was determined by the construction of a graph that illustrates the relationship between the percentage of α-glucosidase activity inhibition and the logarithmic concentration of the salak seed extract. In order to ascertain the specific enzymes that are hindered by the salak seed extract, resulting in the decrease in α-glucosidase activity, a Lineweaver–Burk plot analysis was conducted. The Michaelis–Menten constant (Km), the inhibition constant (Ki), and the maximum metabolic rate (Vmax)were assessed by non-linear regression analysis utilizing the Solver add-in in Microsoft Excel 2010, based on the provided equation [The ICequation :(5)ν=VmaMale ICR mice (30–40 g) were housed in the Animal Center at the Faculty of Pharmacy, Mahidol University at a constant temperature of 25 °C and with a 12-h light-dark cycle. They were given ad libitum access to standard food and water, except when fasting prior to glucose experiments. The experimental protocol was approved by the Animal Ethics Committee of the Faculty of Pharmacy, Mahidol University (permission number: PYR003/2022).2 inhalation. If three or more animals died during the experiment, the limit test was terminated, and the main test was initiated. The LD50 value was considered to be higher than the test dose (2000 mg/kg) if three or more animals survived.The acute toxicity assay of the salak seed extract followed the guidelines of the Organization for Economic Cooperation and Development (OECD), specifically test guidelines 425 (up and down procedure), in accordance with the principles of the limit test [After a single intraperitoneal (i.p.) dose of 120 mg/kg nicotinamide (NA) dissolved in saline, overnight-fasted mice (12 h) were injected i.p. with 180 mg/kg of STZ dissolved in 0.1 M of citrate buffer (pH 4.5). The assessment of diabetes development was conducted after a period of 14–21 days, during which blood glucose levels were measured following an overnight fasting period. Blood samples were obtained from the tail vein, and glucose concentrations were assessed using a glucometer equipped with glucose test strips manufactured by Roche Diabetes Care, Inc., Indianapolis, IN, USA. In our investigation, mice were chosen as subjects if they had fasting blood glucose (FBG) levels beyond 200 mg/dL, indicating the presence of diabetes mellitus (DM) .The anti-hyperglycemic effects of salak seed extract on both normal and STZ-NA-induced DM mice were evaluated by oral glucose tolerance tests (OGTT). A direct toxicity study of salak seed extract has not previously been conducted. However, a previous in vivo study using Wistar rats showed that there were no noticeable signs of toxicity, and no mortality was observed after daily oral administration of 70% ethanol salak seed extract at doses of 0.28 g, 0.56 g, and 1.12 g/kg for 7 days . Further0, C30, C60, C90, and C120 are the FBG concentrations (mg/dL) 0, 30, 60, 90, and 120 min after glucose administration, respectively; t0, t30, t60, t90, and t120 are the times (min) after glucose administration.The methods of Karthikesan et al.  and SatoNormal Control group (NC): Mice in this group received water .Diabetes Mellitus group (DM): Mice with diabetes administered with water.DM + Gli-5: Mice with diabetes administered with 5 mg/kg of glibenclamide (serving as a positive control group).DM + Salak-500: Mice with diabetes administered with 500 mg/kg of salak seed extract.DM + Salak-1000: Mice with diabetes administered with 1000 mg/kg of salak seed extract.The determination of anti-hyperglycemic effects was conducted as described previously . We sele2 inhalation. Blood was collected via cardiac puncture and then centrifuged at 5000 rpm, 4 °C for 8 min to obtain serum and used in the next studies. Serum insulin levels were measured using a mouse insulin ELISA kit , according to the manufacturer’s guidelines.All treatments were conducted once daily via oral gavage for 28 days. During the 28-day treatment period, the body weight and average food intake of the mice were measured daily. The measurement of fasting blood glucose levels was conducted following a 12 h fasting period on days 0, 14, and 28 of the treatment periods. At the end of the trial, the mice passed away by an overdose of COp < 0.05.The data from in vitro studies were presented as the mean ± standard deviation (SD), while the data from in vivo studies were presented as mean ± standard error of mean (SEM). The significant differences were analyzed using one-way ANOVA, followed by post-hoc Tukey’s multiple comparisons. Statistical testing was conducted using SPSS version 18 . The statistical significance level was set at 50 value exceeds 2000 mg/kg. These findings suggest the potential of salak seeds as a valuable component for functional health products and beverages tailored to support the management of postprandial hyperglycemia, which may further contribute to various complications associated with diabetes, such as cardiovascular diseases. We anticipate further research based on our findings, which could help develop additional applications and integration of salak seeds into natural antidiabetic therapies.This study demonstrated the salak seed extract’s capability to mitigate postprandial hyperglycemia in STZ-NA-induced diabetic mice. This in vivo finding was rationalized by in vitro experiments which clarified its effects on glucose-maintaining molecular mechanisms, i.e., α-glucosidase activity and intestinal glucose uptake. Furthermore, our investigation has revealed the antioxidant effects of salak seed extract, notably the inhibition of MDA production as a consequential outcome of lipid peroxidation. The salak seed extract is considered safe, as its LD


Rhizopus microsporusin a 50-year-old man with poorly controlled hypertension, type 2 diabetes, andprolonged use of corticosteroids for the treatment of his reiterative goutyarthritis. In this patient, the use of corticosteroids and poorly controlleddiabetes were considered underlying risk factor for his disseminatedmucormycosis, along with acute renal dysfunction, suggesting the need forclinical suspicion of disseminated pulmonary and renal mucormycosis inhospitalized patients with poorly controlled diabetes and immunocompromisedhost.Rhinocerebral and pulmonary mucormycosis are the main manifestations ofmucormycosis; however, disseminated pulmonary associated with renal mucormycosisis rarely reported. In this paper, we report a rare fatal case of disseminatedpulmonary and renal mucormycosis caused by  The main risk factors of mucormycosis are diabetes mellitus,immunocompromised host, hematological malignancies, neutropenia, solid organtransplantation, autoimmune diseases, multiple injuries, and cortisol use. The increasing prevalence of mucormycosis during the COVID-19 pandemic isbecoming a growing concern. Additionally, the role of diabetes and corticosteroid use an their relationwith COVID-19-related Mucormycosis (CAM) was determined. In recent years, there has been a growing number of reported cases ofdifferent types of mucormycosis, including rhinocerebral, cutaneous, pulmonary,cardiac, gastrointestinal, and disseminated; however, osteomyelitis, peritonitis,endocarditis, and renal infection are rare forms. Species from Rhizopus genus are the most responsible forhigh-mortality mucormycosis. Rhizopus microspores and Rhizopushomothallicus infections are rising in India. The main methods used to diagnose Mucorales infections include direct KOHmount, special staining , as wellas positive culture results from different clinical samples and histopathologicaldemonstration. The Mucorales are usually fast-growing fungi characterized byprimitive coenocytic, broad, and mostly aseptate hyphae. PCR testing and sequencingare reliable diagnostic techniques for Mucorales infections; however, these testsare limited, especially in low-income and middle-income countries.Mucormycosis is a rare life-threatening opportunistic infection characterized byrapid developmentRhizopus microspores in a 50-year-old manwith poorly controlled hypertension, type 2 diabetes, and prolonged use ofcorticosteroids for the treatment of his reiterative gouty arthritis.In this study, we present a rare fatal case of disseminated pulmonary and renalmucormycosis caused by Enterococcus specimen was detected in the blood culture of thepatient’s first hospitalization in another hospital half a month before. Thus,broad-spectrum antibacterial therapy coverage with imipenem and daptomycin was usedwithout determination of a clear infectious etiology after inter-consultation withthe infectious disease team. However, there was no significant clinical improvementwith antibacterial therapy.A 50-year-old rural man presented to the emergency department of the People’sHospital of Guangxi Zhuang Autonomous Region, China, due to frequent cough,sneezing, recurrent fever, and expectoration for one month. Later, he was admittedto the emergency intensive care unit due to acute respiratory failure. He presentedpoorly controlled hypertension and type 2 diabetes, along with long-term prednisoneuse to treat his reiterative gouty arthritis for 10 years due to takinguric-acid-lowering drugs irregularly. The physical examination revealed fever, with40 °C temperature peak, tachycardiac (116 beats/min), and blood pressure at146/79mmHg. Laboratory results showed leukocytosis (lidation. Abdominlidation. He receEnterobacteriaceaeBuganda (meropenem sensitive) was detected in culture from bronchialalveolar lavage fluid (BALF). Therefore, meropenem was used forEnterobacteriaceae Buganda pneumonia. On day 6 of admission,the Gram staining of BALF revealed wide, aseptate hyphae .On day 8 of admission, calcofluor white staining of urine revealed wide, aseptatehyphae  after 24h of incubation for posaconazole(0.5 µg/mL), amphotericin B (2 µg/mL), both suggestive of susceptibility.Consequently, the antimicrobial treatments plan was revised to include meropenem forEnterobacteriaceae Buganda pneumonia, along with intravenousadministration of 100 mg of amphotericin B cholesterol sulfate and nebulization of10 mg twice a day for Rhizopus microspores. Unfortunately, thepatient’s condition deteriorated the following day (day 9) and resulted indeath.On the fourth day of admission, the multidrug-resistant e hyphae . Cotton-e hyphae , which wue stain .Internaehyphae , and Rhi. Rhinocerebral and pulmonary mucormycosis are the main manifestations ofmucormycosis; however, disseminated pulmonary with renal mucormycosis is rarely reported. Early clinical suspicion and accurate identification are crucial formanaging mucormycosis due to its rapid invasion of blood vessels, resulting in highmortality rates. The ability of transferrin to transport iron is inhibited and theconcentration of free iron is reduced in patients with diabetes, which is beneficialfor the growth and reproduction of Mucor. Pulmonary mucormycosis presents obvious dyspnea and hypoxemia. Therefore, clinicians should have a high suspicion of pulmonary mucormycosisin diabetic patients who experience persistent dyspnea and hypoxemia that cannot beattributed to typical pneumonia. Renal mucormycosis is characterized by symptomssuch as fever and flank pain resembling acute pyelonephritis, along with significantimpairment of renal function, including elevated levels of blood creatinine andurea, proteinuria, hematuria, and pyuria.The incidence of mucormycosis have been increasing in patients with diabetes mellitusin recent decade, causing high mortality. The most significant risk factor of CAM was uncontrolled diabetes. Patients with uncontrolled diabetes usually have an inflammatory state,which can be enhanced by activating the antiviral immunity against SARS-CoV-2, whichmay be conducive to secondary infection. The increase in cases of CAM has been associated with continuous use of corticosteroids. A recent report indicated that the incidence rate of mucormycosis wouldincrease even if patients with diabetes were treated with corticosteroids for ashort time. One report has also mentioned that the patients who received a cumulativedose exceeding 600 mg of prednisone or 2-7 g of methylprednisolone are more prone to mucormycosis. Corticosteroids are the most commonly used drugs to treat COVID-19, leadingto a hyperglycemic state in diabetes patients, which provide a fertile soil forMucorales fungi. The coexistence of COVID-19 infection, diabetes, and corticosteroid usecontributed to the disruption of patients’ immune systems, resulting in thedevelopment of invasive mucormycosis. If a patient with COVID-19 is suspected or confirmed to have mucormycosis,all immunosuppressive treatments should be reduced or discontinued based on theclinical condition.During the COVID-19 pandemic, the incidence rate of CAM increased significantly,especially in India. Liposome amphotericin B should be used as a first-line treatment drug andadministered early. Delayed initiation of liposome amphotericin B therapy (>6days after diagnosis) has been associated with doubling mortality rate at three months. Isavuconazole has recently become a first-line alternative for patients whoare either intolerant to amphotericin B or do not respond favorably to it. Moreover, it has been documented that the all-cause mortality ofisavuconazole is similar to that of liposome amphotericin B in a limited number ofpatients as first-line treatment . Some cases of successful treatment of mucormycosis with posaconazole havebeen documented, although it is currently believed that posaconazole can only be consideredas a second-line treatment or salvage treatment. The empirical use of active antifungal drugs for mucormycosis is reasonablein patients with clear risk factors, but routine use of posaconazole formucormycosis prevention remains controversial, which will increase the risk offungal resistance to posaconazole. Furthermore, adjunctive therapy, such as ironchelators, statins, GM-CSF, colistin and cytokines, shows potential to improve outcomes.Combining medical drug treatment with active surgical treatment and optimizing thepatient’s conditions, such as correcting acidosis, correcting hyperglycemia,restoring the white blood cell count, and adjusting immunosuppressants use, cansignificantly improve the survival rate of patientsBriefly, in this case, the primary risk factors for the patient’s disseminatedmucormycosis were the use of corticosteroids and poorly-controlled diabetes. It isuncommon to see disseminated pulmonary with renal mucormycosis. Mortality might bereduced by improved clinical suspicion, early initiation of antifungals, and timelydiagnosis, along with aggressive surgical debridement of necrotic lesions andreversal of underlying immunosuppression.


Brucella genus, transmitted to humans by animals, especially cattle. It rarely involves the nervous system (neurobrucellosis); only a few cases present with hearing loss. We report a case of neurobrucellosis, that presented with bilateral sensorineural hearing loss and mild to moderate persistent headache. To the best of our knowledge, this is the first well-documented case from Nepal.Brucellosis is a zoonotic disease, caused by a Gram-negative coccobacillus of The patient was a 40-year-old Asian male shepherd from the western mountainous region of Nepal who came to the emergency department of Manipal Teaching Hospital, Pokhara in May, 2018 and did a follow-up for 6 months. He presented with high-grade fever, profuse sweating, headache, myalgia, and bilateral sensorineural hearing loss. His history of consuming raw milk of cattle, symptoms including persistent mild to moderate headache, bilateral hearing loss, and serological findings were suggestive of neurobrucellosis. Following treatment, the symptoms improved, including the complete recovery of hearing loss.Hearing loss may be the manifestation of neurobrucellosis. Physicians should know about such presentations in brucella endemic areas. Brucellosis is a chronic granulomatous disease that usually presents with vague symptoms such as fever, diaphoresis, malaise, myalgia, arthralgia, and headache, as well as some atypical symptoms because of multiple organ involvement , 2. BrucNeurobrucellosis is seen in up to 10% of cases of brucellosis –7. NeuroWe report a case of a 40-year-old Asian male shepherd who presented with the chief complaint of recurrent high-grade fever with an evening rise in temperature of 1 month duration associated with profuse sweating, mild to moderate headache, myalgia, and anorexia. He had experienced decreased hearing in both ears for the last 10 days. He noticed his clothes became too loose for him. He did not report diabetes mellitus, hypertension, or any other chronic illness. The patient had a high fever with a 103° F recorded temperature, tachycardia with a pulse rate of 110/min, low body mass index (BMI) of 18, normal blood pressure, and a respiratory rate of 18/min. Physical examination revealed splenomegaly (14 cm), but no hepatomegaly or enlarged lymph nodes. Whisper, Rinne, and Weber tests were performed and sensorineural hearing loss was suggested by deafness to whispering sound, air conduction better than bone conduction, and no lateralization. Pure tone audiometry showed bilateral mild-moderate sensorineural hearing loss  (34 mm in 1st hour). Tuberculin skin test for tuberculosis, K39 serology for leishmaniasis, widal test for typhoid, thick and thin smear for plasmodium were performed and all test results were negative. Bone marrow aspiration cytology and trephine biopsy with H&E stain showed reactive marrow with non-caseating granulomatous inflammation Fig. . After tConsidering the consumption of cattle’s raw milk, endemicity of brucella in Nepal, neurological findings unexplainable otherwise, and positive CSF brucella agglutination test, we diagnosed the case as neurobrucellosis. We treated him with doxycycline (100 mg twice a day for 6 months), and streptomycin injections (1 g/day for 21 days). Follow-up after three months showed complete resolution of symptoms, except hearing loss that improved after completion of therapy at 6 months. To the best of our knowledge, this is the first case report of neurobrucellosis from Nepal.Brucellosis has a wide geographical distribution with over 500,000 human cases per year worldwide , 10, 11.Although there are no definite criteria for Neurobrucellosis , some arThere is no consensus on the dose, type of antibiotics, and duration of treatment in Neurobrucellosis , 21, 22.Brucellosis is often misdiagnosed as malaria, tuberculosis, and typhoid fever which may cause mistreatment and under-reporting in Nepal. Our case met the diagnostic criteria of Neurobrucellosis; therefore, this is the first case reported from Nepal, with sensorineural hearing loss and headache as a manifestation of the disease. Knowing this rare possibility, clinicians should include brucella tests in patients presenting with hearing loss, in brucella endemic regions as early diagnosis and treatment have better outcomes. This case should affect the practice of physicians who interact with sensorineural hearing loss in brucella endemic areas.Since Brucellosis is a zoonotic disease, we suggest clinicians collaborate with veterinarians who are involved in identification and treatment of infected animals, as this will help prevent further transmission to humans. This is also the core of the “One Health Policy” by World Health Organization (WHO) in controlling zoonotic diseases . The govNeuroimaging could not be performed due to a shortage of economic resources.


Age-related hearing loss (ARHL) signifies the bilateral, symmetrical, sensorineural hearing loss that commonly occurs in elderly individuals. Several studies have suggested a higher risk of dementia among patients diagnosed with ARHL. Although the precise causal association between ARHL and cognitive decline remains unclear, ARHL has been recognized as one of the most significant factors that can be modified to reduce the risk of developing dementia potentially. Mild cognitive impairment (MCI) typically serves as the initial stage in the transition from normal cognitive function to dementia. Consequently, the objective of our randomized controlled trial (RCT) is to further investigate whether the use of hearing aids can enhance cognitive function in older adults diagnosed with ARHL and MCI.This study is a parallel-arm, randomized controlled trial conducted at multiple centers in Shanghai, China. We aim to enlist a total of 688 older adults (age ≥ 60) diagnosed with moderate-to-severe ARHL and MCI from our four research centers. Participants will be assigned randomly to either the hearing aid fitting group or the health education group using block randomization with varying block sizes. Audiometry, cognitive function assessments, and other relevant data will be collected at baseline, as well as at 6, 12, and 24 months post-intervention by audiologists and trained researchers. The primary outcome of our study is the rate of progression to dementia among the two groups of participants. Additionally, various evaluations will be conducted to measure hearing improvement and changes in cognitive function. Apart from the final study results, we also plan to conduct an interim analysis using data from 12-month follow-up.In recent years, there has been a notable lack of randomized controlled trials (RCTs) investigating the possible causal relationship between hearing fitting and the improvement of cognitive function. Our findings may demonstrate that hearing rehabilitation can be a valuable tool in managing ARHL and preventing cognitive decline, which will contribute to the development of a comprehensive framework for the prevention and control of cognitive decline.Chinese Clinical Trial Registry chictr.org.cn ChiCTR2000036139. Registered on 21 August 2020.The online version contains supplementary material available at 10.1186/s13063-023-07813-z. Age-related hearing loss, also known as presbycusis, is a complex syndrome caused by the cumulative effects of aging on the auditory system, characterized by age-related, progressive, bilaterally symmetrical, and sensorineural hearing loss, which usually occurs at high frequencies in older adults firstly . AccordiARHL induces a huge financial burden, which brings additional outlays of 1 trillion dollars a year . HoweverIt has been recognized for decades that older adults with hearing loss had a higher risk of dementia , 19, in Nonetheless, the causality between ARHL and cognitive decline is not clear now . AccordiMCI, occurring in the early process of cognitive development from normal to dementia, usually refers to a cognitive disorder which is significantly more severe than cognitive decline in normal aging, while patients retain most of the ability to perform independent activities of daily living , 35. ItsSince we pay more attention to their connection, the hearing intervention has been considered to be a potential therapy for cognitive decline . ConsideThis multicenter, parallel-arm randomized controlled trial (RCT) study will be conducted by four tertiary referral clinics : Ninth People’s Hospital, Sixth People’s Hospital, Ruijin Hospital, and Mental Health Center. The study protocol is drafted following the Standard Protocol Items Recommendations for Interventional Trials (SPIRIT) statement  at the outpatient clinics of our study centers, those who have been informed about the study through online advertisements, and participants who are already enrolled in the cohort study of the CHOICE project. Since our investigation focuses on the development of dementia, which predominantly occurs in the late stages of life, we will specifically consider individuals who are aged 60 or older in order to ensure the external validity of our findings.Upon providing signed informed consent, participants will be required to complete a questionnaire that covers their basic information, which is necessary for the research. Additionally, participants will undergo auditory tests and evaluations of cognitive function to fulfill the criteria established for the RCT study.Age ≥ 60Postlingual hearing lossReceived education for at least 6 yearsSensorineural hearing loss35 ≤ PTA  < 80 dB HLMini-mental State Examination ≤ 26Geriatric Depression Scale ≤ 10Activity of Daily Living Scale ≤ 17Used hearing aids within a yearInability to complete cognitive assessmentsConductive hearing lossUnwilling to wear hearing aids dailyHad neurosyphilis or hypothyroidismConsidering that the subjects in our experiment will be aware of whether they have received the hearing aid fitting, we will not be utilizing the double-blind method. In order to minimize potential bias and ensure a fair distribution of participants, we will randomly assign subjects from the outpatients and community cohort to either the intervention group or the control group in a 1:1 ratio. To prevent predictability, we will employ block randomization using variable block sizes . This wiAfter randomization, every participant allocated to the hearing aid fitting group will get the binaural and behind-the-ear hearing aids, Widex-Evoke E-FA100 & E-FP50, with a full set of auxiliary accessories, purchased by Ninth People’s Hospital and distributed to each sub-center. Our professional audiologist will make adjustments for patients until they can hear what others say with a few noisy environmental sounds, which will make participants willing to learn the usage of hearing aids and wear them in their daily lives. In addition, some of the subjects with a long history of hearing loss may lead to poor effect of hearing aids. We will provide some suggestions on hearing rehabilitation for them, including reading newspapers out and talking to their families frequently. Patients are required to wear hearing aids for more than 10 h per day. The time of wearing hearing aids will be recorded by a system; thus, we can remind subjects. Furthermore, participants will maintain their hearing aids if needed and make regular adjustments to their hearing aids with the help of an audiologist.The patients in the health education group will receive periodic health education from researchers as part of the study, including valuable guidance on hearing health, such as the importance of noise reduction, avoidance of ototoxic drugs, and development of healthy ear care habits.Furthermore, we will make it clear to the participants that those who are allocated to the intervention group will benefit from using hearing aids free of charge for the rest of their lives, while those who are assigned to the control group will receive an extra allowance at the end of the trial period. Besides, in the hearing aids group, we will use the system in hearing aids to record the average duration of hearing aid use, and for those who do not meet the requirement, we will take measures such as the commissioning of hearing aids and online supervision. All participants will get subsidies for the cost of transportation for every follow-up visit.In the follow-up stage, the patients of the two groups will be recalled to study centers to perform assessments of auditory tests and cognitive evaluations at 6, 12, and 24 months after the intervention. Researchers will contact those who are near the date of the follow-up visit and make an appointment with them in advance.Subjects are not allowed to participate in another trial which may influence the results during the study.The assessment of the following outcomes will take place during the screening, baseline measurements, and follow-up visits at 6, 12, and 24 months after the intervention   at a souTympanometry is often used to assess the conditions of the middle ear, including the presence of fluid, changes in the internal structure, and the ear canal volume. It can be classified as type A, meaning the normal situation of the middle ear; type B, indicating that there is a significant abnormality in it; and type C, suggesting the negative pressure of the middle ear .A listening test platform from INTERACOUSTICS A/S will be used to measure the tympanograms of both ears. Patients with type B will be ineligible for our study, which indicates the presence of fluid in the middle ear with the consequences of inflammation mainly, implying the secretory otitis media and potential conductive hearing loss .Cognitive abilities, such as working memory and executive ability, play important roles in speech discrimination . After tCAP is usually used in assessing the auditory condition in patients with hearing loss and the process of recovery after hearing intervention . The oriIOI-HA is an international standard self-report questionnaire developed to quantify the satisfaction of hearing aid users and the improvement of their lives by the application of hearing aids . It consCognitive function encompasses various domains, including basic sensory and perceptual processes, as well as advanced executive function and cognitive control abilities . To condMMSE consists of temporal and spatial localization, word retelling, calculation, language use, comprehension, and basic motor skills , which hCDR is widely used in the measurement of the patient’s cognitive impairment, which consists of evaluations in six domains . The score of 0 in CDR represents normal, while 0.5 represents MCI or suspected dementia and 1, 2, and 3 represent mild, moderate, and severe dementia .ADAS-Cog is usually performed to evaluate the cognitive and behavioral functions in patients impaired by Alzheimer’s disease (AD) and is considered to be the gold standard for evaluating the efficacy of dementia treatment . The scaThe minimal clinically relevant change (MCRC) on the ADAS-Cog for patients with AD is usually among 3 to 5 points. 4 points are recommended by the FDA as the appropriate MCRC for clinical trials of patients .Geriatric Depression Scale (GDS)Depression is usually suggested being a risk factor and a prodromal symptom of dementia . So we wActivity of Daily Living Scale (ADL)ADL-14 consists of 14 questions, involving the whole set of behaviors of people in daily life, devised for evaluating the activity functional state of individuals systematically and personalized by asking their family numbers. The higher score on ADL-14 indicates greater functional disability .Functional activities questionnaire (FAQ)FAQ is commonly used to assess the functional limitations in older adults . The queShort Form-12 (SF-12)The SF-12 is the shortened alternative to the SF-36, containing 12 subsets. It is often used to evaluate the living quality of participants with a focus of whole physical and mental health outcomes , 70.According to the previous study, cumulative blood pressure is one of the risk factors in dementia with negative cognitive effects . And higNo foreseeable adverse events related to hearing aid fitting are expected to occur in the study. However, in the event of any unexpected adverse events, they will be documented and reported following the standard operating procedures set forth by the Ethics Committee of Shanghai Ninth People’s Hospital.Participants have the right to withdraw from the study at any time without providing a reason. Any participants who are lost to follow-up will be recorded, and an intention-to-treat analysis will be performed exclusively for these individuals.Blinding is not possible in this study due to the nature of the intervention for participants or researchers, but we intend to blind the data analyzers to reduce bias in the data analysis. Moreover, we will employ several strategies to mitigate bias from both researchers and participants. Firstly, prior to their involvement in the CHOICE project, researchers will receive standardized training to ensure consistent data collection and analysis. Additionally, we will utilize standardized measurement tools and quality control procedures to monitor data accuracy and reliability. Furthermore, we will ensure that participants in both the intervention and control groups are well-informed about the potential benefits and drawbacks of their respective treatments, aiming to minimize biases in their perceptions of therapeutic effects.α was set at 0.05, 1 – β = 0.8, and hearing aid fitting and health education group were set at 1:1; thus, 292 cases in each group were calculated. Considering the 15% of drop-out rate, we designed 344 cases in each group, and a total of 688 cases. Shanghai Ninth People’s Hospital will be responsible for 516 cases, Shanghai Sixth People’s Hospital for 103 cases, and Shanghai Ruijin Hospital for 69 cases. Furthermore, Shanghai Mental Health Center will provide training about cognitive evaluations for our researchers and recommend eligible patients for our study.To detect the clinically relevant difference in the dementia rate after the hearing aid fitting compared with the health education group, the sample size of this study is calculated as follows. Our pilot study results showed that 3 of 30 patients with ARHL and MCI wearing hearing aids developed dementia within a year. Literature reported that 18% of the elderly with MCI progressed to dementia every year . In concAll research centers will use the paper Case Report Form designed for the study to record the original data. Then, they will be input into the Shen-kang Clinical Research Integration Platform system by trained researchers from Shanghai Ninth People’s Hospital. The database is password-protected, which can only be accessed by specified researchers. All changes made in the database will be logged. The final data set will be only available to authors for research.The research centers will utilize the designated paper Case Report Form (CRF), specifically designed for this study, to record the original data. Subsequently, trained researchers from Shanghai Ninth People’s Hospital will input the data into the Shen-kang Clinical Research Integration Platform system. Access to the database is protected by a password and limited to authorized researchers. Any modifications made within the database will be meticulously logged. The final dataset will solely be accessible to the authors for research purposes.t-test will be used to calculate the differences between the control and intervention groups. For within-group comparisons, paired t-tests will be used for continuous measures. Non-parametric tests will be used to compare data that do not follow a normal distribution. The differences in outcomes between groups will be determined by calculating the means and 95% confidence intervals. A P value < 0.05 will be considered statistically significant. Additionally, mixed effects models, COX regression, and multifactorial linear regression will be utilized to further explore the association between hearing aid fittings and cognitive improvement.The mean and standard deviations, or the median and quartiles, will be used to describe the baseline features of both the hearing aid fitting group and the health education group, depending on the normality of the data. The unpaired Intention-to-treat analysis will be conducted on all subjects enrolled in the group. Additionally, a per-protocol analysis will be performed on subjects with high compliance, specifically those who have worn hearing aids for the duration required by the experiment. To handle missing data, multiple imputation will be utilized. Complete case analyses will be conducted as a sensitivity analysis. It is important to note that all analyses will be performed on an intention-to-treat basis.We will perform an interim analysis when all subjects complete the 12-month follow-up visits using the baseline data and follow-up data collected at 6 and 12 months after the interventions, and if the results of the interim analysis are positive, we will consider terminating the study.Researchers, including audiologists, cognitive evaluators, and data collectors, will be required to ensure full compliance with specifications and pass the consistency test during the assessment process. Additionally, 5% of the subjects will be randomly selected for audio recording of the entire cognitive evaluation process, which will be submitted to the Shanghai Mental Health Center for quality control.An external data monitoring committee will monitor the study. The committee will be independent of the sponsor and researcher in the study and have no competing interests. The committee will have an audiologist, a psychiatrist, and a statistician, who will review the provisional data when the completion of data collection for the first year. The implementation of projects will be tracked and reviewed by the Ethics Committee of Ninth of Shanghai Ninth People’s Hospital every year.All subjects will participate in the study voluntarily, and the senior researchers in our project team, such as Dr. Hao Wu, Dr. Haibo Shi, Dr. Mingliang Xiang, Dr. Xia Li, Dr. Ying Chen, Dr. Wen Lu, Dr. Bin Ye, Dr. Yuan Fang, will obtain their written consent forms from each individual prior to their enrolment in the group. It is expected that both groups of subjects will derive benefits from the study. Additionally, we will inform all participants about any new information that may influence their willingness to participate in the experiment. The trial will adhere to the criteria and principles outlined in the Declaration of Helsinki and has received approval from the Ethics Committee of Shanghai Ninth People’s Hospital (project ID: ChiCTR2000036139) . We willThe entirety of the research-related data, including original data and records, quality control files, and software used for data storage and follow-up analysis, will be limited to access by the study team only. The statistical analysis and processing of the collected data will be conducted exclusively by designated researchers. The identity and contact information of all participants will be strictly confidential. Prior to performing statistical analysis, the data will be anonymized. Ultimately, the research findings will be published in reputable scientific journals.One focus of ARHL studies is the causal relationship between hearing loss and cognitive decline. In recent years, research has shown that hearing loss is the most significant modifiable factor in preventing dementia. However, currently, there is limited research on hearing interventions that improve the cognitive function of ARHL patients, particularly in randomized controlled trials. The scarcity of studies may be attributed to the prolonged development process of dementia or the absence of clear evaluative indicators for cognitive function, which adds to the challenge of conducting RCT trials. The ACHIEVE project, the sole published large-scale randomized controlled trial, focused on whether the use of hearing aids reduces the incidence of dementia among cognitively normal individuals over a 3-year follow-up period and yielded negative findings .According to previous research, about 16% of older adults have MCI, and individuals with MCI have a higher likelihood of progressing to dementia at a faster rate compared to those with normal cognitive function. Furthermore, cognitive impairment is often accompanied by hearing loss in older adults, with nearly half experiencing moderate hearing loss or worse . TherefoIn conclusion, in addition to monitoring the development of dementia, we intend to investigate the effectiveness of hearing intervention on various cognitive domains in patients with ARHL and MCI. We anticipate that hearing rehabilitation can be proven to be an effective therapeutic approach for cognitive recovery, thus enhancing our ability to prevent and manage ARHL.Recruitment for this randomized controlled trial (RCT) study commenced in 2021, with the first patient enrolled on September 28, 2021. Currently, subject enrollment is still in progress (November 2023). It is anticipated that the recruitment, screening, random allocation, and baseline evaluation of all participants will be finalized by 2023. Following the completion of the 1-year follow-up for all subjects, the gathered data will be utilized for an interim analysis.Protocol Version 3: 18 November 2023.The study is funded by the Clinical Research Plan of SHDC (SHDC2020CR1044B to HW), Natural Science Foundation of Shanghai (23ZR1437100 to YC), National Natural Science Foundation of China (82371141 to YC), and Biobank Project of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (YBKA202205 to YC). All research funding for our study is handled by the research department of Ninth People’s Hospital, so we provide the contact information of the hospital research funding administrator, Ms. Qing Li, with the contact email kjc5325@vip.163.com. Moreover, in the study, the sponsor is not involved in the research design, data collection, data management, data analysis and interpretation, writing of the manuscript, and publication of the paper. They will not have ultimate authority over any of these activities.Additional file 1. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents.


SLC26A4 gene and includes non-syndromic and syndromic types. This study aimed to identify genetic defects in a Chinese patient with non-syndromic enlarged vestibular aqueduct (NSEVA) and to investigate the impact of variants on the severity of non-syndromic enlarged vestibular aqueduct. A male patient with NSEVA, aged approximately 6 years, was recruited for this study. The clinical characteristics and results of auxiliary examinations, including laboratory and imaging examinations, were collected, and 127 common hereditary deafness genes were detected by chip capture high-throughput sequencing. Protein structure predictions, the potential impact of mutations, and multiple sequence alignments were analyzed in silico. Compound heterozygote mutations c.1523_1528delinsAC (p.Thr508Asnfs*3) and c.422T>C (p.Phe141Ser) in the SLC26A4 gene were identified. The novel frameshift mutation c.1523_1528delinsAC produces a severely truncated pendrin protein, and c.422T>C has been suggested to be a disease-causing mutation. Therefore, this study demonstrates that the novel mutation c.1523_1528delinsAC in compound heterozygosity with c.422T>C in the SLC26A4 gene is likely to be the cause of NSEVA. Cochlear implants are the preferred treatment modality for patients with NSEVA and severe-to-profound sensorineural hearing loss Genetic counseling and prenatal diagnosis are essential for early diagnosis. These findings expand the mutational spectrum of SLC26A4 and improve our understanding of the molecular mechanisms underlying NSEVA.Enlarged vestibular aqueduct is an autosomal genetic disease mainly caused by mutations in the  SLC26A4 gene, including Pendred’s syndrome (PS) and non-syndromic EVA (NSEVA). Notably, some patients with EVA have only one mutation or lack detectable mutations. Patients with different mutations also exhibit high phenotypic heterogeneity. High intrafamily phenotypic variability occurs among individuals with PS, and the typical goiter phenotype of PS is variably penetrant; some patients may not display symptoms until adolescence (Enlarged vestibular aqueduct (EVA) is an autosomal recessive disease characterized by severe-to-profound sensorineural hearing loss (SNHL) and widening of the vestibular aqueduct . EVA is lescence . TherefoSLC26A4 gene is located on the human autosome 7q22.3, contains 21 exons, and is closely associated with EVA occurrence and development. The pendrin protein encoded by SLC26A4 plays a significant role in endolymphatic fluid resorption, acid-base balance, and proper function of the inner ear  and c.1523_1528delinsAC (p.Thr508Asn fs*3), in the A roughly 6-year-old boy presented to our hospital on 21 April 2019 due to “hearing loss for 2 year.” The patient was diagnosed with profound bilateral sensorineural deafness at the age of 4 years. The child could communicate normally after wearing hearing aids in both ears and undergoing speech rehabilitation training, but he could only hear voices within 3 meters; therefore, he was admitted for cochlear implantation surgery.Physical examination showed a temperature of 36.8°C, pulse rate of 76 beats/min, respiratory rate of 20 beats/min, blood pressure of 110/75 mmHg, weight of 19 kg, and height of 114 cm. The child did not have mastoid tenderness or external ear deformity and had an unobstructed external auditory canal. The tympanic membrane was normal, and the tympanic curve was unobstructed. He had normal growth and development, was born full-term naturally, with a birth weight of 3,000 g, and passed the initial hearing screening 3 days after birth. He had a history of pneumonia but denied any exposure to ototoxic drugs, ear trauma, ear surgery, long-term noise exposure, early pregnancy infection, goiter, or diabetes mellitus. The parents were physically healthy and other members of the family had no hearing loss. The child underwent cochlear implantation in the left ear and follow-up showed good hearing test results.A 6-year-old boy with profound sensorineural deafness was recruited and detailed history questioning, physical examinations, laboratory tests, and imaging examinations were performed. His parents were also enrolled in the present study. Blood-EDTA samples were obtained and subsequently used for DNA extraction and downstream sequencing analysis.High-throughput sequencing was performed by the NGS platform of the BGISEQ  using a hearing impairment panel. Briefly, genomic DNA was randomly fragmented and used for the library. The DNA in the coding region of the target genes and the adjacent splicing region was captured and enriched by the chip and sequenced through the BGISEQ platform. A total of 127 genetic deafness-related genes were analyzed.Genomic DNA was extracted from 1 mL EDTA-anticoagulated peripheral blood of all subjects using a TIANamp blood DNA kit , according to the manufacturer’s protocol. DNA quality was quantified using a NanoDrop 2000c spectrophotometer . PCR was performed in a SimpliAmp™ PCR thermocycler  with a total volume of 20 μL, including 1×DNA Polymerase, 1×Taq Buffer, 2.5 mM of dNTP mixture, 0.5 µM of forward and reverse primers, and 50 ng of DNA template. The PCR conditions were as follows: 95°C for 5 min; 38 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min. PCR products were assessed by 1.5% agarose gelelectrophoresis and directly sequenced with an ABI 3730xl DNA analyzer.https://swissmodel.expasy.org/) program. The potential impact of the mutations was predicted by MutationTaster, Mutation Assessor, PolyPhen-2, and FATHMM software. The alignment of pendrin protein sequences in multiple species  was performed using ClustalX2 software.Three-dimensional structure molecular modeling of wild-type and mutant pendrin was carried out using the SWISS-MODEL (The auditory brainstem response (ABR) test showed an increase in the response threshold and that the response wave was elicited at 100.0 dBnHL  and 85 dBnHL for the left and right ears, respectively. The pure tone audiometry was significantly above the threshold value. Abnormal results were detected in binaural distortion product otoacoustic emission and acoustic reflex tests. Craniocerebral computed tomography (CT) and magnetic resonance imaging (MRI) showed bilateral enlarged vestibular aqueducts and endolymph sac effusion, respectively . The patSLC26A4, MT-RNR1, and MT-TS1  mutation was predicted to cause a frameshift and emergence of an early stop codon, resulting in the production of a severely truncated pendrin protein  patients with bilateral EVA carried biallelic SLC26A4 mutations  are the primary methods for hearing recovery in patients with EVA. Conventional hearing aids can provide good hearing rehabilitation in patients with mild-to-severe SNHL. However, CI is the preferred treatment as hearing loss progresses because hearing aids cannot meet their needs, and the early use of hearing aids may help patients better adapt to CI . A systelization . Additioutations . In the SLC26A4 mutations should undergo genetic counseling, and prenatal diagnosis is feasible if necessary. In cases of significant hearing loss, especially congenital SNHL cases associated with inner ear malformations, SLC26A4 variants and other common pathogen-related variants must be considered in molecular examinations (Previous studies, including this case study, have revealed several important points. First, early detection of hearing loss is beneficial for hearing development and the wellbeing of children , and neoinations . High-thinations .SLC26A4 gene is likely to be the cause of NSEVA. Patients with double-site mutations exhibit severe sensorineural deafness and EVA. Early diagnosis, timely intervention, and genetic counseling are crucial for reducing disease progression and adverse effects. The findings of our study expand the mutational spectrum of SLC26A4 associated with NSEVA and may be helpful for the prevention and early intervention of family based NSEVA.This study demonstrates that the novel mutation c.1523_1528delinsAC in compound heterozygosity with c.422T > C in the