Sudden hearing loss is an otorhinolaryngological emergency that often leads to severe damage to the auditory and vestibular function. The vestibular evoked myogenic potential is a test that allows a noninvasive evaluation of the otolithic system function and vestibulospinal and vestibulo-ocular pathways.To evaluate the importance of vestibular evoked myogenic potential in determining the prognosis of patients with sudden hearing loss.A search for articles published up to December 2018 was performed in the PubMed, Cochrane, VHL and LILACS databases using MeSH descriptors. Retrospective and prospective articles were included containing cervical or ocular vestibular evoked myogenic potential in sudden hearing loss patients and information on associated vertigo and/or dizziness.Sixteen of 62 initially selected articles met the inclusion criteria and were analyzed. Regarding the methodology of the evaluated studies, 8 studies were prospective, six were retrospective, one contained part of the data from a retrospective analysis and another part from a prospective analysis, and one study was cross-sectional. A total of 872 patients were evaluated  with a mean age of 51.26 years. Four hundred and twenty-six patients (50.35%) had vertigo and/or dizziness associated with sudden hearing loss. The cervical vestibular evoked myogenic potential was performed in all studies, but only seven assessed the ocular vestibular evoked myogenic potential. The cervical vestibular evoked myogenic potential showed alterations in 38.65% of 846 evaluated ears, whereas ocular vestibular evoked myogenic potential showed alterations in 47.88% of 368 evaluated ears. The hearing recovery rate was analyzed by 8 articles, with 63.4% of 410 evaluated ears showing hearing recovery.The studies suggest that the assessment of the vestibular system using vestibular evoked myogenic potential seems to be important in the prognosis of sudden hearing loss. For better follow-up of patients with sudden hearing loss, the emphasis should not be limited to the cochlea, but also include the diagnosis and treatment of vestibular abnormalities, regardless of the presence of vertigo. Sudden hearing loss (SHL) is an otorhinolaryngological emergency defined as a hearing loss of at least 30 dB, at three consecutive audiometric frequencies, of sudden onset within 72 h.The pathophysiology of SHL is not yet fully established. Some associations with viral infections, vascular disorders, and autoimmune diseases have been reported in the literature.In addition to damage to the hearing function, SHL can lead to changes in vestibular function.Several medications have been investigated for the treatment of SHL. However, systemic corticosteroids have been recommended as the drug of choice.Some factors related to prognosis have been described, such as age, presence of vertigo, severity, and audiometric pattern of hearing loss, VEMP, evoked auditory brainstem response (ABR), and otoacoustic emissions (OAE).VEMP is a short-latency evoked potential that allows the noninvasive assessment of the otolithic system function and vestibulospinal and vestibulo-ocular pathways.In some studies, a greater number of patients with profound hearing loss have been found to have abnormal VEMP results.Finally, this review aims to evaluate the importance of VEMP in SHL, summarizing the available data on the alterations of this test in the prognosis of patients with this disease.A systematic review on VEMP in SHL was performed using MeSH descriptors in the PubMed, Cochrane, VHL and LILACS databases for studies published until December 2018. After combining specific keywords , articles written in English, Portuguese and Spanish were selected manually. An additional bibliographic research was performed to provide specific information regarding VEMP and SHL.The following eligibility criteria were used for inclusion in the analysis of this review: retrospective or prospective evaluation through cervical or ocular VEMP in patients with sudden hearing loss and information on associated vestibular symptoms. Case reports, book chapters, systematic reviews, and studies that did not provide sufficient information for the analysis in this project were excluded.The necessary information and data were extracted from the selected studies and quantified using a standardized procedure. The characteristics of each study were evaluated, such as year of publication, study design, age, gender, number of assessed patients, laterality, presence of vertigo and/or dizziness, moment of VEMP performance, abnormalities in cVEMP or oVEMP , auditory recovery and moment of the final audiometric assessment. The hearing recovery criteria was considered as any improvement from 10 decibels on the average of at least four frequencies of the initial tonal audiometry, regardless of the classification  used in the studies.The assessment of quality and risk of bias of the analyzed studies was performed using the Agency for health care research and quality (AHRQ) checklist.According to the abovementioned criteria, 62 articles were initially selected. Of these, 25 were excluded by the abstract, 2 because they were systematic reviews, 1 because it was a book chapter and 8 articles because they were written in Chinese. Of the 26 remaining articles, 10 were excluded after reading the full article, as they did not provide sufficient information to verify the methodological quality required for inclusion in this study. Finally, 16 articles met the necessary criteria for the final review .Figure 1,12,Clinical data and complementary examinations of the 16 selected articles are listed in 6Sudden hearing loss is a relatively common event in otorhinolaryngology and has been extensively studied since its first description in the literature.Findings about prognosis and vestibular involvement in SHL are uncertain, which motivated this review. The VEMP is a complementary test, able to evaluate the function of otolithic organs. The cVEMP mainly evaluates the saccule and inferior vestibular nerve, while oVEMP evaluates the utricle and superior vestibular nerve. Therefore, we decided to analyze the importance of VEMP in the prognostic assessment of patients with SHL based on articles published in the literature.The VEMP examination can be performed using click sound or tone burst stimuli in dB SPL (decibel sound pressure level) or dB HL (decibel hearing level).Of the seven articles that simultaneously evaluated both exams (cVEMP and oVEMP), four showed a higher number of patients with changes in oVEMP than in the cVEMP,The auditory recovery of SHL patients and associated factors was also analyzed in this review. Of the reviewed articles, six showed that altered VEMP in SHL patients was associated with poor hearing recovery.15Some limitations of the analyzed articles are not being multicentric or case-control or cohort studies; sample size; no data masking and lack of a prolonged follow-up. The investigation of sudden hearing loss with several tests, including vestibular evoked myogenic potential , Video Head Impulse Test (vHIT) and videonystagmography could better clarify the pathophysiology and extent (the structures affected in the inner ear) of the disease, in addition to being useful as a prognostic assessment tool for auditory recovery in patients with SHL.The studies suggest that the vestibular system assessment using VEMP seems to be important in the prognosis of sudden hearing loss. For better follow-up of patients with sudden hearing loss, the emphasis should not be limited to the cochlea, but also include the diagnosis and treatment of vestibular alterations, regardless of the presence of vertigo.The authors declare no conflicts of interest.


Gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs) are a rare, highly malignant subset of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). However, how to predict the prognosis of GEP-NECs by clinical features is still under study. This study aims to establish and validate a nomogram model of overall survival (OS) in patients with GEP-NECs for predicting their prognosis.We selected patients diagnosed with GEP-NECs from the Surveillance, Epidemiology, and End Results (SEER) database and two Chinese hospitals. After randomization, we divided the data in the SEER database into the train cohort and the test cohort at a ratio of 7:3 and used the Chinese cohort as the validation cohort. The Cox univariate and multivariate analyses were performed to incorporate statistically significant variables into the nomogram model. We then established a nomogram and validated it by concordance index (C-index), calibration curve, receiver operating characteristic (ROC) curve, the area under the curve (AUC), and the decision curve analysis (DCA) curve.We calculated the nomogram C-index as 0.797 with a 95% confidence interval (95% CI) of 0.783–0.815 in the train cohort, 0.816 (95% CI: 0.794–0.833) in the test cohort and 0.801 (95% CI: 0.784–0.827) in the validation cohort. Then, we plotted the calibration curves and ROC curves, and AUCs were obtained to verify the specificity and sensitivity of the model, with 1-, 3- and 5-year AUCs of 0.776, 0.768, and 0.770, respectively, in the train cohort; 0.794, 0.808, and 0.799 in the test cohort; 0.922, 0.925, and 0.947 in the validation cohort. The calibration curve and DCA curves also indicated that this nomogram model had good clinical benefits.We established the OS nomogram model of GEP-NEC patients, including variables of age, race, sex, tumor site, tumor grade, and TNM stage. This model has good fitting, high sensitivity and specificity, and good clinical benefits. Neuroendocrine carcinomas (NECs) are a rare, highly malignant subgroup of neuroendocrine neoplasms (NENs), while the gastroenteropancreatic system is one of the most common areas from which NECs can originate , 2. WithAt present, few studies focus only on GEP-NECs, while more studies concentrate on gastroenteropancreatic neuroendocrine tumors (GEP-NETs), another subtype of gastroenteropancreatic NENs (GEP-NENs) that are less malignant and have more cases and a wider range. It is generally believed that the factors affecting the prognosis of GEP-NETs mainly include the patient’s age, tumor grade, pathological stage, and primary tumor site , 11–13. For this purpose, we retrieved and collected the data of patients with GEP-NECs from the Surveillance, Epidemiology, and End Results (SEER) database and two Chinese hospitals and constructed and validated a nomogram based on clinicopathological information.We reviewed patients diagnosed with GEP-NECs between 1988 and 2019 from the SEER database, which was established in the United States to provide first-hand information for clinical work worldwide , 15. AllTo better verify the applicability of the constructed nomogram model in Chinese patients, we reviewed patients of the Second Hospital of Jilin University and China-Japan Union Hospital of Jilin University between 2010 and 2018. The screening criteria were consistent with those patients from the SEER database, which are described above. Finally, 70 patients with GEP-NECs were selected as the validation cohort. The data filtering process is shown in While the prognostic factors of GEP-NECs are still uncertain, prognostic factors of GEP-NETs were used as variables to be included in the nomogram model. Existing studies have shown that older age, male sex, high tumor stage, and low tumor differentiation are markers of poor tumor prognosis, and tumors originating in the pancreas have the worst prognosis –23. TherWe used IBM SPSS Statistic to perform univariate and multivariate Cox regression models in the train cohort to determine variables that would be output for the establishment of the nomogram model. Once we obtained these variables, we used R software 4.2.1 to build a nomogram model. Then, the test and validation cohorts were used to evaluate the newly established nomogram. The comparison between nomogram prediction and actual observation was assessed by the concordance index (C-index) and the calibration curve. The receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to evaluate the sensitivity and specificity of the model. In addition, we plotted the decision curve analysis (DCA) curve of this nomogram model to verify the clinical benefits. All analyses were completed with R software 4.2.1 and IBM SPSS software, and the analysis was statistically significant only when P<0.05.According to the inclusion and exclusion criteria, a total of 4251 cases in the SEER database were eventually included in this study, of which 2976 were assigned to the train cohort and 1275 were randomly assigned to the test cohort. Among all patients, 46.72% were younger than 60 years old, 53.28% were older than 60 years old, 53.94% were male, and 46.06% were female; 11.79% were black, 76.55% were white, and 11.67% were other races; 32.96% had large intestine cancer, 20.63% had small intestine cancer, 38.37% had pancreatic cancer, and 8.05% had stomach cancer. No significant differences were found between the train and test cohorts in each contained variable. Furthermore, we also selected 70 cases from the two qualified Chinese hospitals as the validation cohort, among which 52.86% were younger than 60 years old, 47.14% were older than 60 years old, 70% were male, and 30% were female. Since these cases were from the colorectal surgery departments of two hospitals in China, none of the patients were white or black, and the primary tumor site was in the large intestine. All results are shown in The results of univariate and multivariate analyses are shown in The validation process was carried out internally and externally using the C-index and calibration curves as validation tools. Specifically, the C-index of the OS nomogram was 0.797, with a 95% confidence interval (95% CI) of 0.783–0.815 in the train cohort and 0.816 (95% CI: 0.794–0.833) in the test cohort, as shown in The same process was also performed in the validation cohort. The C-index was 0.801 (95% CI: 0.784-0.827) in the nomogram model Figure 3According to the above description, we constructed a nomogram model based on age, race, sex, tumor site, tumor grade, and TNM stage, and the validation showed that the model fitting effect was good. Regardless of the train cohort, the test cohort, or the validation cohort, the C-index was greater than 0.7, and the slope of the calibration curve was close to 1. This indicates that the constructed nomogram model has a good prediction effect and is close to the actual situation. For the ROC curve, the AUCs were all greater than 0.7, showing higher accuracy and specificity of the model. Meanwhile, the DCA curves showed good clinical benefit in all three cohorts. The above verification process proves that the nomogram model constructed has a good fitting effect, and all the introduced variables can be used as prognostic factors for GEP-NECs, which is that male sex, older age, high tumor stage and grade are markers of poor tumor prognosis. The results are also similar to existing research , which aAt present, due to the rarity of GEP-NECs, few studies have focused on the prognosis of GEP-NECs alone and the construction of a prognosis prediction model, while papers have concentrated more on the construction of prediction models using larger datasets of GEP-NETs. The study by Zi-Han Xu et al. included GEP-NETs in the SEER database and constructed a nomogram model based on age, sex, race, marital status, tumor grade and stage, radiotherapy, chemotherapy, and surgery, obtaining the C-indexes for OS prediction in the nomogram as 0.893  and 0.880 , respectively, in the train cohort and validation cohort, with the AUCs of the nomogram predicting the 3-year and 5-year OS rates as 0.908 and 0.893, respectively, which shows an effective function of prediction . The varCurrently, with the continuous expansion of genomic information, research using genomic information to construct a prognostic model is emerging. The study by Nobuyoshi Takizawa et al. proved the similarity between colorectal neuroendocrine carcinomas and adenocarcinoma by gene sequencing analysis . ShinichBased on the above, it may be possible to integrate clinicopathological information and genomic information into model construction in the future. Through this model, we can correlate clinicopathological information and genetic information that affect the prognosis of diseases to achieve accurate control of diseases and treat diseases from the root.To our knowledge, this study is one of the largest and most recent studies focusing on GEP-NECs, providing comprehensive epidemiological and survival data for GEP-NECs, constructing a complete nomogram model, and yielding good predictive results. However, there are some limitations to our study. For example, as Ki-67 and the mitotic index, which are absent in the SEER database, are very important for tumor grading , these fWe established the OS nomogram model of GEP-NEC patients, including variables of age, race, sex, tumor site, tumor grade, and TNM stage. This model has good fitting, high sensitivity and specificity, and good clinical benefits.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.JC, YL, and HS designed the study. JC and KX performed the research. YL and FR analyzed the data. JC and YL wrote the paper. BL and HS revised the manuscript for final submission. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Beauveria bassiana, a fungal pathogen that only infects insects, to test their susceptibility to infection. The tests with bed bugs revealed that they were highly susceptible to fungal infections, no matter how we exposed them to the pathogen. The German cockroaches were only infected by fungi through certain routes of exposure. Fungal pathogens have the potential to control bed bugs but will require additional research and innovative technologies to be effective against cockroaches.Bed bugs and German cockroaches have adapted to thrive in human structures. In the present study, we use different techniques to expose bed bugs and German cockroaches to Cimex lectularius) and omnivorous German cockroach (Blattella germanica) are highly successful indoors, but have evolved different physiological and behavioral adaptations to cope with the human-built environment, some of which also reduce the efficacy of fungal biopesticides. In order to gain greater insight into the host barriers that prevent or constrain fungal infection in bed bugs and German cockroaches, we tested different doses of Beauveria bassiana GHA through surface contact, topical application, feeding, and injection. Bed bugs were generally more susceptible to infection by B. bassiana with the mode of delivery having a significant impact on infectivity. The German cockroach was highly resilient to infection, requiring high doses of fungal conidia (>8.8 × 104) delivered by injection into the hemocoel to cause mortality. Mortality occurred much faster in both insect species after exposure to surfaces dusted with dry conidia than surfaces treated with conidia suspended in water or oil. These findings highlight the importance of developing innovative delivery techniques to enhance fungal entomopathogens against bed bugs and cockroaches.The ability of a fungal entomopathogen to infect an insect depends on a variety of factors, including strain, host, and environmental conditions. Similarly, an insect’s ability to prevent fungal infection is dependent on its biology, environment, and evolutionary history. Synanthropic pests have adapted to thrive in the indoor environment, yet they arose from divergent evolutionary lineages and occupy different feeding guilds. The hematophagous bed bug ( Metarhizium anisopliae (Sorokin), Beauveria bassiana , and Isaria fumorosea (Wize) have a wider host range than other fungi that infect insects and produce large quantities of conidia [Several times throughout their evolutionary history, fungi have evolved various strategies and adaptations to use living insects as resources . Fungi i conidia . Some hy conidia ,3, althoMusca domestica (Linnaeus) larvae, which live and feed in septic decaying materials, produce chitosan, which is anti-bacterial and can suppress the growth of several different fungi in vitro [Aphis gossypii (Glover), behaviorally avoid plants that are infected with entomopathogenic fungi [Coccinella septempunctata (Linnaeus), avoid prey insects that are infected with Beauveria bassiana [Trybliographa rapae (Westwood) avoid entomopathogen-infected hosts [Potential insect hosts have evolved various physiological and behavioral adaptations to counter or prevent fungal infections. These adaptations can be linked to their mode of feeding and food source, habitat, and evolutionary history. For example, in vitro . Phytophic fungi . Generalbassiana . Similared hosts .Cimex lectularius (Linnaeus) have significantly lower allelic diversity than that in bat-associated populations of the same species, suggesting an ancestral genetic bottleneck in the human-associated lineage [kdr mutations in response to exposure to DDT and pyrethroids [Blattella germanica (Linnaeus) cannot fly, likely as an adaptation to the indoor environment, whereas its close relative Blattella asahinai (Mizukubo) lives outdoors and is capable of flight [Indoor environments are harsh, and only a few species, considered pests, have adapted to exploit human-built niches . Some of lineage . The humethroids ,12, wherf flight . SurprisBeauveria bassiana strain GHA on two human-associated host species with disparate life histories: C. lectularius and B. germanica. By exposing bed bugs and cockroaches to conidia through different routes, including surface contact, topical treatment, ingestion, and injection, we sought to broadly identify and compare their barriers to fungal infection. These barriers include the exterior cuticle , the interior cuticle, digestive tract, and peritrophic membrane (ingestion), and the immunological barriers (injection). We also investigated whether suspending the conidia in oil or keeping them dry increased the infectivity of B. bassiana. The findings of this study provide important insights into the optimal routes of exposure to fungal entomopathogens for biocontrol of bed bugs and German cockroaches.Therefore, our overall goal was to compare the infectivity of the fungal entomopathogen B. germanica  and C. lectularius (Harold Harlan strain) were maintained at 27 ± 1 °C, 12 L:12 D, and approximately 50% RH. These strains are reference insecticide-susceptible strains which were collected in 1947 and 1973, respectively. All experiments were conducted at 26 ± 1 °C, 12 L:12 D, and approximately 50% RH. The optimal growth temperature for B. bassiana GHA is 25 °C [Colonies of is 25 °C . CockroaBed bug colonies were fed defibrinated rabbit blood in an artificial feeding system . BrieflyB. bassiana GHA (henceforth referred to as B. bassiana) conidia were provided by Dr. Nina Jenkins. The conidia were transferred to sterile containers and then suspended in sterile distilled water with 0.01% Tween-80 . Suspensions of different conidia concentrations were then created by serial dilution of the stock suspension. The conidia concentration of the suspensions was confirmed using an Improved Neubauer hemocytometer. Viability of the conidia was confirmed to be >98% by plating suspensions of known concentrations onto Petri dishes containing ~20 mL of sterile Sabouraud dextrose agar . The conidia counts were confirmed for each of the experiments described below by plating an aliquot of the counted suspension on 20 mL of sterile Sabouraud dextrose agar in 90-mm plastic petri dishes.Pure B. bassiana while bypassing the host cuticular barrier, we injected conidia suspended in 1X PBS  into the hemocoel of the insects. Ten cockroaches were used per replicate. Individuals were incapacitated by chilling (8 °C for 10 min) in a refrigerator before being placed dorsal side down onto a filter paper in a chilled 90-mm Petri dish and injecting 1 µL of a known concentration of conidia (4.4 × 102 to 1.3 × 106 conidia per insect) suspended in sterile 1X PBS using a 32-gauge beveled needle and 10-µL syringe . The injections were performed by hand, and entry was made between the 6th and 7th abdominal sternites away from the insect midline. After injection, the insects were transferred to a glass jar (60 × 100 mm) containing a pellet of rodent chow and a glass water tube stoppered with cotton. The control cockroaches were injected with either 1 µL of sterile 1X PBS or 1 µL of heat-killed (autoclaved) 1.3 × 106 conidia.To assess the maximal efficacy of 2 to 8.8 × 105 conidia per insect). The control bed bugs were injected with either 0.1 µL of sterile 1X PBS or 0.1 µL of deactivated (autoclaved) 8.8 × 105 conidia.For the bed bug injection assays, 10 adult males were used per replicate. The bugs were restrained dorsal side down on double-sided sticky tape that was attached to a glass microscope slide. We used an autoinjector  with a needle drawn from a glass capillary  to inject the bed bugs with a 0.1-µL suspension of conidia in 1X PBS  and checked daily for sporulation of the fungi. The same timeline for mortality scoring and mycosis induction was used for all the experiments described below.Three replicates per concentration (n = 30) were conducted with the cockroaches and bed bugs. After the injections, the insects were placed in a temperature-controlled room as described above, and mortality was scored every 24 h for 14 days. To confirm that mortality was caused by B. bassiana on cockroaches through the digestive tract, we adapted the method of Wada-Katsumata et al. [6 conidia) was pipetted onto a plastic microscope slide (22 × 22 mm), forming a stable liquid droplet. The microscope slide was transferred to a glass Petri dish housing a single cockroach, and the dish was covered to exclude light. The cockroaches were given 2 h to ingest the liquid droplet. The cockroaches that fully consumed the droplet (> 90%) were transferred to jars, and mortality was scored daily as described above. The control insects were fed 4 µL of a 1 M sucrose solution, and the positive controls were fed 80 µg of boric acid (2%) in 1 M sucrose + 0.01% Tween-80 to confirm that this was an effective method to deliver a toxicant to cockroaches.To assess the infectivity of a et al. . Briefly3 to 4.4 × 106 conidia per insect) was calculated based on the assumption that each male consumed ~4 µL of blood [The bed bugs were fed blood supplemented with conidia. Briefly, suspensions of conidia were prepared in sterile distilled water with 0.01% Tween-80. The conidia suspension (30 µL) was then added to 2.97 mL of rabbit blood . Groups of 30 male bed bugs were then placed into feeding vials as described above and allowed to feed for 30 min. The dose of conidia ingested by each male . Suspensions of conidia in oil (from 8.8 × 104 to 4.4 × 106 conidia per insect) were prepared by concentrating or diluting a stock oil suspension of B. bassiana conidia  with oil (Conidiotec). Next, the cockroaches and bed bugs were incapacitated using the refrigerator and placed dorsal side down onto a Petri dish before 1 µL of the conidia in water or oil was applied to the metathoracic area of each insect. The cockroaches were transferred to jars, and the bed bugs were transferred to Petri dishes. Each treatment group was replicated three times for both insect species. The control groups consisted of treatment with 1 µL of sterile distilled water with 0.01% Tween-80 or 1 µL of oil.To compare the ability of B. bassiana to infect cockroaches and bed bugs through contact with a treated surface, 90-mm filter papers  were sprayed using an airbrush sprayer (Conidiotec) with either oil or B. bassiana conidia suspended in oil . All suspensions were thoroughly agitated before use, and the airbrush was held horizontally to spray the filter papers. To ensure that the entire area of the filter papers was treated, two passes were made over them  at a rate of 30 cm/sec. The airbrush was kept 10 cm from the filter papers. The treated filter papers were allowed to dry for 24 h and then placed in the lids of the 90-mm Petri dishes so that the base could form a tight seal and prevent escape. The insects were then cold-anesthetized and transferred to the Petri dishes. The cockroaches were provided with a water tube and rodent food that had the bottom covered so it did not contact the treated filter paper. The pellet was not tested for B. bassiana contamination, but no visible sporulation was observed from the rodent chow. The lids were then secured using labeling tape, and the Petri dishes were incubated at the conditions described above. The treatments and controls (oil) were replicated two and five times for the bed bugs and cockroaches, respectively.To assess the ability of 6 conidia/cm2. After drying for 24 h, the treated filter papers were placed in 100 × 10 mm glass Petri dish lids so the insects only had access to the treated filter paper by using the base as the top (~0.5 cm was uncovered). Dry conidia  were placed on a 90-mm filter paper in a glass Petri dish and evenly spread using a fine paint brush. The insects were anesthetized and placed in the Petri dishes with food and water as described above. Each treatment group was replicated 3 times with 10 insects per replicate.Because the conidia that were suspended in water or oil were ineffective on the German cockroaches, we compared the infectivity of conidia suspended in water or oil with dry conidia that were deposited on a surface with both insects. We treated the 90-mm filter papers with conidia suspended in either water or oil with an airbrush sprayer as described above, resulting in 1.3 × 10The mortality of the insects exposed to different concentrations of conidia during each 14-day experiment was analyzed using Kaplan–Meier survival analysis in SPSS 27 . Different treatments and control groups were then compared in a pairwise manner using a log-rank test. Insects that survived beyond the 14-day period were censored to indicate survival. Because survival analysis assumes equal probabilities of individuals leaving the study , we addeB. bassiana GHA on bed bugs and German cockroaches by delivering conidia to the insects using various means. We also assayed the effects of an oil formulation of conidia to determine if oil enhanced the infectivity of B. bassiana. Bed bug survivorship was significantly affected by B. bassiana, with all five routes of exposure  . Even at lower doses (4.4 × 102 to 4.4 × 104 conidia per insect), we achieved high mortality (76.6–83.3%). It is notable, however, that the pairwise comparisons showed no significant differences among these three doses . The overall high infectivity of B. bassiana in bed bugs after bypassing the cuticular barrier suggests that they are highly susceptible to B. bassiana.We used two independent control injections—PBS and autoclaved conidia—to assess the effects of the injection on the bed bugs and cockroaches. In both species, injected PBS or autoclaved conidia resulted in minimal  mortality A,B, and  0.0001) A. The moB. bassiana was much less infective against cockroaches than bed bugs (4) caused low mortality, but at higher doses, the cockroaches experienced high mortality, which mostly occurred 1–2 days after injection. Thus, there was a clear separation of survivorship curves above and below 4.4 × 104 conidia . There was a generally broad statistical overlap among the high-dose treatments, but two treatments were significantly different . Only one of the doses (1.3 × 106 conidia) caused 100% mortality.Although injection was also the most effective route of exposure for German cockroaches, bed bugs B. Low doB. bassiana conidia were effective when fed to bed bugs , with high doses causing 100% mortality by day 3  (2) resulted in > 50% survivorship estimates after 14 days, whereas higher doses were much more effective at killing the bed bugs .The arisons) C. Simila6 conidia per cockroach failed to kill any cockroaches in the feeding assays. These results indicate that B. bassiana conidia are ineffective at infecting cockroaches through the digestive tract.We validated our feeding method for the cockroaches by incorporating 80 µg of boric acid into the sugar solution. All cockroaches died by day 8, as expected D. Howevep < 0.001). Topical application of ≤ 8.8 × 105 conidia caused 10–53.3% mortality (MSTs 11.2–13.2 days), whereas 1.3 × 106 conidia, the highest dose we tested, caused 93.3 ± 3.3% mortality 14 days after treatment   E. Bed buB. bassiana dose of 1.3 × 106 conidia suspended in water caused no mortality in the cockroaches   G. HoweveB. bassiana conidia on the cockroaches. Topical treatment with ≤1.3 × 106 conidia had minimal effects which were not significantly different from those of oil alone . However, topical application of 4.4 × 106 conidia in oil caused significant mortality , with 70% killed within 14 days . Thus, it appears that oil slightly enhanced the efficacy of B. bassiana against German cockroaches.In contrast to the bed bugs, the oil by itself caused only 10% mortality in the male German cockroaches on day 1, and 90% survived to day 14 H. Therefp < 0.001). Concentrations ≥8.8 × 106 conidia/cm2 killed 100% of the bed bugs during the 14-day exposure period, with significantly lower MST estimates at higher concentrations . These results demonstrate that the B. bassiana conidia suspended in oil and sprayed onto a filter paper surface were effective against bed bugs by contact.We applied conidia suspended in oil to filter papers, and 24 h later, bed bugs or cockroaches were placed on the filter papers. All the bed bugs survived for 14 days on the water-treated control filter papers, and 23.3% experienced mortality on the oil-treated control filter papers I. ContacB. bassiana. The oil alone, when applied to the filter papers, caused no mortality in the cockroaches, and only 20% mortality occurred after 14 days of continuous contact with the highest concentration of 8.8 × 106 conidia/cm2 . The mean survival time at this concentration was 12.5 ± 0.4 days, significantly lower than those at all other concentrations .In contrast to the bed bugs, the German cockroaches J were rep < 0.001). The bed bugs exposed to dry conidia experienced a mean survival time of 5.5 ± 0.3 days, representing significantly faster mortality than that in the bed bugs exposed to surfaces sprayed with conidia suspended in water or oil  (6 conidia/cm2) resulted in similar survivorship , with mean survival times of 6.8 ± 0.3 days and 7.4 ± 0.6 days, respectively, and 100% mortality by day 14. The two control treatments had similar survivorship , with no mortality in the water control group and 6.6% mortality in the oil control group.We found significant differences among the three treatment groups (dry conidia applied directly to the filter paper surface and conidia suspended in water or oil and sprayed onto the filter paper) and two control groups (water or oil sprayed on filter papers)  A. Equivap < 0.001) (6 conidia/cm2) suspended in water or oil demonstrated that both treatments were ineffective against the cockroaches, with only 3.3% mortality in cockroaches exposed to conidia sprayed in water and 0% mortality in cockroaches exposed to conidia sprayed in oil, consistent with previous findings (p > 0.55 for other comparisons). In contrast, the cockroaches exposed to dry conidia reached 66% mortality by the end of the 14-day experiment, with a mean survival time of 7.6 ± 0.9 days, which was significantly different from those of the other treatments . These results demonstrate striking differences in the infectivity of B. bassiana conidia applied in dry form or as suspensions in water or oil. The conidia were much less infective when sprayed in a liquid suspension than when applied in dust form. Although this loss of effectiveness was evident against both bed bugs and German cockroaches, it was particularly severe with the cockroaches.We found significant differences in the survivorship of German cockroaches in the three treatments and two control groups  B. A compfindings J. These B. bassiana in all the above-mentioned bioassays. There was no evidence of sporulation under high humidity conditions in the bed bugs that were killed by the oil alone.We confirmed mycosis in 100% of the cadavers of insects exposed to Beauveria bassiana GHA, which has a broad host range, on two sympatric synanthropic pests with divergent life histories: the bed bug (Cimex lectularius) and the German cockroach (Blattella germanica). By using different modes of exposure, we sought to determine the extent to which various barriers prevent fungal entomopathogen infection that could potentially be overcome with further studies. Although some modes of exposure were more effective than others, we found that, overall, the bed bugs were susceptible to B. bassiana through all the tested routes of exposure. These findings are consistent with previous research using contact assays with the same B. bassiana strain [B. bassiana, except when conidia were introduced directly into the hemocoel by injection or when exposed to dry aerial conidia that were not sprayed in a liquid suspension. Below, we consider different factors that might contribute to the susceptibility of bed bugs and resilience of cockroaches to B. bassiana.The infectivity of host insects by fungal entomopathogens depends on a variety of factors, including environmental conditions, characteristics related to the biology of the host, and features of the pathogen itself. In this study, we compared the infectivity of the fungal entomopathogen a strain ,19,20. OC. lectularius and Cimex hemipterus have adapted to feed on human blood and infest residential settings [Bed bugs may not have evolved strong defense mechanisms against fungal entomopathogens because of their unique evolutionary history and current ecology. Most cimicids are associated with and primarily feed upon bats and birds, but settings ,22. Humasettings ,22. Althsettings ,24, bed Conversely, cockroaches occupy a wide variety of habitats and feed on an array of resources, such as decaying organic material in leaf litter, leaves, algae and other epiphylls on leaf surfaces, and decaying wood ,26. MostConidiobolus coronatus Batko (Costantin)-containing solutions, revealed that some free fatty acids were inhibitory, whereas others were susceptible to fungal degradation [Alternaria solani, Fusarium oxysporum, and Colletotrichum lagenarium [The insect cuticle represents the first barrier against environmental stressors, desiccation, and entomopathogen infection ,31. Featradation . Gutierrgenarium . These lgenarium ,37, also4 and its complete resilience to 1.3 × 106 conidia applied directly to the cuticle. In contrast, the bed bug cuticle must remain pliable to accommodate vast expansion of the abdomen with large blood meals, and because it has an ample supply of water in each blood meal, it appears to invest much less in the production of cuticular lipids. The collateral effect may be the susceptibility of bed bugs to cuticular penetration by fungal pathogens when exposed through different routes, as was observed in this study.Moreover, whereas the bed bug cuticle is approximately 10 µm thick , the thiE)-2-hexenal and (E)-2-octenal, suppress M. anisolpliae growth in vitro, and (E)-2-hexenal at >200 µg was also antagonistic to B. bassiana [E)-2-hexenal than bed bugs normally produce [Bed bugs produce short-chain aldehydes as alarm and aggregation pheromones . Two aldbassiana . However produce , so it rBed bugs and cockroaches evolved divergent self-grooming strategies that could influence entomopathogen infectivity. During grooming, hemipterans concentrate external particles on the tarsi and tibia and then dislodge them by rubbing against a surface . This grIn contrast, German cockroaches groom extensively to improve chemoreception and courtship success, and this process also removes environmental contaminants and microorganisms from the exterior of the insect ,45,46. GCryptocercus, a subsocial cockroach whose ancestors may have given rise to termites, lives inside and feeds upon wood and has antifungal compounds in salivary secretions and on the cuticle that are used in self- and allogrooming of nestmates [Cryptocercus hindgut also has abundant β-1,3-glucanase activity, and their fecal pellets have antifungal properties [Cryptocercus help prevent pathogen outbreaks in conditions that are favorable to fungal growth. Termites also possess similar adaptations. In simulated colony set-ups of the subterranean Reticulitermes flavipes, they were able to inhibit germination of Metarhizium anisopliae conidia [Zootermopsis angusticollis produces multiple β-1,3 glucanases, which are antifungal [Zootermopsis angusticollis, which are embedded in the nest structure, also have antifungal properties and prevent fungal outbreaks [Blattella germanica may share some of these antifungal adaptations [In support of this hypothesis, estmates . The Cryoperties . Both of conidia . Termite conidia . The con conidia . The guttifungal . The fecutbreaks . Becauseptations ,49.B. bassiana through feeding. Notably, hemipterans lack a defined peritrophic membrane [Bacillus thuringiensis israelensis and Pseudomonas entomophilia [Beauveria conidia and other entomopathogens are less able to prevent infection.Bed bugs are highly susceptible to ingested insecticides ,58 and bmembrane ,61, a chmembrane . Hemiptemembrane ,61. Humomophilia . However6 conidia).The German cockroach, on the other hand, produces a well-organized peritrophic membrane whose components are upregulated by food intake . It is aB. bassiana conidia, as in the present study. The bed bug genome includes genes with putative immune functions , but the diversity of immune genes is sparse compared with other insect genomes. Both recognition proteins and antimicrobial peptides are particularly under-represented in bed bugs [B. bassiana is further supported by our observation that similar doses were nearly equally effective by injection and ingestion. For example, a minimal dose of 4.4 × 102 conidia was slightly more effective by injection, but a dose of 8.8 × 104 was equally effective by injection and ingestion, causing 100% mortality by day 4.The final layer of defense against fungal conidia is the hemolymph and the associated immune system. The insect hemolymph is a nutrient-rich environment for fungal conidia to germinate, but it also contains hemocytes capable of encapsulation and phagocytosis of pathogens and antimicrobial peptides . Bed bugbed bugs . The oveB. bassiana conidia that was effective against the German cockroaches. We observed a sharply delineated threshold, where doses <4.4 × 104 conidia were ineffective while higher doses killed >85% of the treated cockroaches. These observations suggest that the higher doses of conidia can overcome the immune system of the cockroach. The hemolymph of B. germanica contains a variety of immunocytes and other hemocytes [Aspergillus [B. germanica revealed 39 AMP genes, which had not initially been annotated and likely play an important role in microbial immunity [B. germanica thrives.Injection into the hemocoel was the only route of delivery of suspended emocytes . For exaergillus . Severalergillus . Recent immunity . Other nC. lectularius, but B. bassiana has been consistently effective against bed bugs. Experiments with B. germanica, however, have yielded mixed results, with both high and low efficacy of different B. bassiana strains. We suspect that such disparate results may be due to differences in methodology, the deployment of conidia, and the virulence of the fungal strains. For example, unlike our findings, other researchers found B. bassiana to be effective against B. germanica in feeding experiments [B. bassiana are only marginally effective against B. germanica [B. bassiana strain GHA was relatively ineffective against B. germanica by all practical routes of exposure. On the other hand, M. anisopliae has been proven to be more effective against the German cockroach by both contact and ingestion [M. anisopliae may be more effective against cockroaches, since they use only one type of insecticidal secondary metabolite, destruxin, to induce host mortality [B. bassiana relies upon the cumulative effect of secondary metabolites [Sparse research has been conducted on the effectiveness of other fungal entomopathogens against eriments ,74. Thesermanica ,75, and ngestion ,77. M. aortality ,79, wherabolites ,81.Culicoides nubeculosus (Meigen) and Hylobius abietis (Linnaeus) than “wet conidia” that were suspended in water with a surfactant [B. bassiana conidia suspended in water, oil, or in dust form revealed that only the dry conidia caused mortality in B. germanica. These findings are consistent with previous work, showing that dry aerial conidia of M. anisopliae caused high mortality in B. germanica [B. bassiana conidia caused high mortality in bed bugs, although the dry conidia were most effective. Overall, the results with cockroaches and bed bugs suggest that B. bassiana conidia are somehow affected by being suspended in water or oil, making it more difficult for conidia to be dislodged from the surface, adhere to the cuticle, or penetrate the cuticle. Aerial conidia have a negative electrostatic charge [B. germanica and C. lectularius are exposed to dry and previously wet conidia.These disparities led us to consider how conidia are formulated and delivered to the insects. The use of water or oil to suspend conidia, as well as surfactants such as Triton X-100 or Tween-80, can affect the adhesive properties of conidia and reduce the infectivity of entomopathogens ,82. Thusrfactant ,82. Our ermanica . In contc charge . Therefoc charge  and bed c charge  allow foB. bassiana in oil improved the efficacy of conidia against B. germanica, but we could not attain more than 70% mortality with a high dose of 4.4 × 106 conidia in oil. In bed bugs, however, the oil by itself caused 100% mortality within a day, precluding any inferences about its role in facilitating the infectivity of conidia. Oils have suffocating activity in sufficient volumes [Aside from selecting more infective strains, two broad strategies to improve the efficacy of entomopathogens include (1) increasing penetration of the cuticle and gut and (2) protecting conidia from the harsh environments where the host insect lives. Petroleum-based oils appear to do both by increasing infectivity at low humidity , protect volumes , but we B. germanica [M. anisopliae against B. germanica, but the mechanism remains unknown [B. germanica [C. lectularius [Isaria fumosorosea in the cockroach Periplaneta americana [Boric acid also synergizes the infectivity of entomopathogens when applied to or fed to ermanica  by disruermanica . Talc po unknown . Insectiermanica  and C. ltularius , likely tularius . Interestularius , also prmericana . It is cB. bassiana GHA conidia by exposure via surface contact, topical application, feeding, and injection. The high mortality of bed bugs in the feeding experiments was likely due to the lack of a well-defined peritrophic membrane. The German cockroach was exceptionally resilient to infection by B. bassiana GHA, likely due to adaptive mechanisms that prevent infections, such as physical and chemical cuticular barriers, self-grooming behavior, high turnover of its peritrophic membrane, and several expanded pathways of antimicrobial and immune response proteins. Oil formulations enhanced the infectivity of B. bassiana GHA conidia in B. germanica, but the oil itself was toxic to bed bugs, precluding an analysis of the effects of conidia in oil. Finally, dry aerial conidia caused greater mortality in both bed bugs and cockroaches than conidia suspended in either water or oil. These findings confirm that fungal entomopathogens should be considered for bed bug control and highlight the importance of discovering strategies that enhance the infectivity of fungal entomopathogens against B. germanica. Bed bugs are generally susceptible to


Gut samples of 34 patients  with metastatic GEP-NENs  were analyzed. Twenty-nine patients had well differentiated GEP-neuroendocrine tumors (GEP-NETs),  and five patients had poorly differentiated GEP-neuroendocrine carcinomas (GEP-NECs). Patients with GEP-NENs had significantly decreased bacterial species and increased fungi  compared to controls. Patients with GEP-NECs had significantly enriched populations of specific bacteria and fungi  compared to those with GEP-NETs . In addition, higher grade GEP-NETs were associated with significantly higher Bacteroides fragilis (p = 0.022), and Eggerthella lenta (p = 0.00018) species compared to lower grade tumors. There were substantial differences associated with dietary habits and therapeutic responses. This is the first study to analyze the role of the microbiome environment in patients with GEP-NENs. There were significant differences between GEP-NETs and GEP-NECs, supporting the role of the gut microbiome in the pathogenesis of these two distinct entities.Gut microbiome balance plays a key role in human health and maintains gut barrier integrity. Dysbiosis, referring to impaired gut microbiome, is linked to a variety of diseases, including cancers, through modulation of the inflammatory process. Most studies concentrated on adenocarcinoma of different sites with very limited information on gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). In this study, we have analyzed the gut microbiome  in patients with metastatic GEP-NENs. Fecal samples were collected and compared with matched healthy control samples using logistic regression distances utilizing R package MatchIt . We examined differences in microbiome profiles between GEP-NENs and control samples using small subunit (SSU) rRNA (16S), ITS1, ITS4 genomic regions for their ability to accurately characterize bacterial and fungal communities. We correlated the results with different behavioral and dietary habits, and tumor features including differentiation, grade, primary site, and therapeutic response. All tests are two-sided and  Neuroendocrine neoplasms (NENs) are a diverse group of neoplasms that arise from endocrine cells throughout the body; a significant proportion originate in the gastrointestinal tract . A SurveThe gut microbiome, composed of diverse communities of bacteria, fungi, viruses, archaea, and protozoa, plays a key role in several metabolic functions and influences host immunity and general health . The co-All participants were recruited at the University Hospitals Seidman Cancer Center, Cleveland, Ohio. Written informed consent was obtained from all participants in this study before collection of the samples. Recruitment of study participants was performed according to protocol (#20200733) approved by the Human Subjects Institution Review Board (IRB) of Case Western Reserve University, Cleveland (OH), and UH Cleveland Medical Center, Cleveland (OH). We collected fecal samples from 40 GEP-NEN patients and 40 age- and sex-matched healthy controls. The healthy control cohort consisted of subjects who had screening colonoscopies and were exposed to the same environment and share similar dietary habits. Both patients and healthy subjects were American inhabitants and most of them consumed a western diet. Race was classified as White , Black/African American, Asian, and Native American/Native Alaskan, etc. Current dietary habits, intake of antibiotics in the last 6 months before enrollment, intake of probiotics or vitamins, smoking status, alcohol intake in addition to age, gender, and ethnicity were all collected from GEP-NEN participants. Summary of demographic information of subjects enrolled in the study are shown in TM CultureSwab), including a user guide. The samples were immediately placed in previously prepared fast prep tubes  containing 500 μL glass beads  and 1 mL ASL™ lysis buffer (Qiagen DNA Extraction Kit) and were transported to the laboratory where assays were conducted. Samples were kept in a −20 °C freezer until they were analyzed.Fecal samples were collected using a specific kit consisting of a ready-to-use package  was used to extract DNA via the manufacturer’s instructions. Samples were pelleted by centrifugation and re-suspended in 1 mL of InhibitEX lysis Buffer. Fecal swabs were incubated for 1 h at 75 °C and mixed in Fastprep 96 2× for 300 s at 1800 RPM. Equal volumes of 100% ethanol and lysate were mixed and passed over HiBind DNA Mini Columns . The DNA extract was eluted into 50 µL of molecular grade water. The quality and purity of the isolated genomic DNA was assessed by gel electrophoresis and quantified using a Qubit 2.0 instrument and Qubit dsDNA HS Assay . DNA samples were stored at −20 °C.Amplification of the 16S and 5.8S rRNA genes was performed using 16S-515 (5′-GGA CTA CCA GGG TAT CTA ATC CTG-3′) and 16S -804 (5′-(TCC TAC GGG AGG CAG CAG T-3′) and ITS1 (5′-(TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) primers, respectively ,13. PCR Amplicon libraries were cleaned and barcoded followed by emulsion PCR using Ion Torrent S5 Prime workflow . Bacterial 16S rRNA and fungal ITS amplicons of equal volumes were pooled, cleaned using AMPure XP beads  and then exposed to end repair enzyme for 20 min at RT. Following an additional AMPure clean up, ligation reactions were performed at 25 °C for 30 min using an Ion Torrent P1 with a unique barcoded ′A′ adaptor per pooled sample. AMPure removal of residual adaptors was performed, and samples were concentrated to 1/4 volume for 1 h under heat and vacuum. Separate barcoded samples were pooled in equal amounts (10 μL) and size selected for the anticipated 16S and ITS range (200–800 bp) using Pippin Prep . Library amplification was performed for seven cycles and quantified using a StepOne qPCR instrument prior to proper dilution to 300 pM before adding the samples into IonSphere templating reactions (Ion Chef). Library sequencing was performed using an Ion Torrent S5 sequencer (ThermoFisher Scientific). Barcoded and sorted samples were analyzed using our custom pipeline based on Greengenes V13_8 and UNITE database V7.2 designed for the taxonomic classification of 16SrRNA and ITS sequences, respectively. Sequencing reads were clustered into operational taxonomic units , described by community metrics, and classified within the Qiime 1.8 taxonomy bioinformatics pipeline.Raw 16s and ITS data of the bacterial read counts was obtained and was loaded to R version 4.0.3., sample meta data was obtained, and, using R package microbiome 1.12.0 and phyloseq 1.34.0 a phyloseq object containing a read count matrix (species level identification), a taxonomic table (kingdom to species) and metadata table were made. When dealing with both 16s and ITS data, each is initially handled separately and then combined after the read counts are normalized and transformed to Relative abundance.Prior to creating the phyloseq object for the 16s and ITS data, the taxonomic table is cleaned to remove species and phyla that are annotated as “unidentified” or lacking identification at a species level annotated as an empty cell. This step allows us to ensure only species that are identifiable are being utilized in the analysis.The main filtering step performed is on the total read counts per sample, where a cutoff of 500 read counts in the 16s data is used as a minimum requirement. This brings down the total number of samples from 40 to 34 samples. Additionally, samples with missing metadata annotations were also removed.Composition bar graphs were generated on 16s and ITS relative abundance data separately after aggregating the data to the phyla level, and filtering on phyla prevalence of 25% amongst all samples. This allows for an overview of differences in the core Phyla between samples.Ordinate analysis/PCA was performed on microbiome and mycobiome data separately, after aggregating data to the phyla level as a means to reduce complexity. The data are then filtered on prevalence of 75% amongst all samples. Ordinate analysis is performed using the “ordinate” function part of microbiome R package version 1.12.0 with method used as “MDS”  and distances using “Bray–Curtis” method.Venn diagrams were generated using the eulerr R package version 6.1.0, using the 16s and ITS species level relative abundance. The core microbiome within each group is determined using a prevalence of 50% within samples in each group.16s and ITS relative abundance data are first filtered on species with a prevalence of 50% within all samples, and then the respective phyloseq objects are merged. Wilcoxon-rank-sum nonparametric test is used to test for significantly different species between the groups being compared, and the fold change is calculated using mean relative abundance of a species within each group using the fold change function from R package tools version 3.9.2. For clinical features that contained more than 2 groups, a Kruskal–Wallis test is used to test for significance in difference between groups. Additionally, Spearman correlation is used to test for significant correlation between clinical features and species within samples. Box plots using ggplot2 R package were generated for all the significantly different species.We studied the stool microbiota of 34 patients with GEP-NENs, as well as bacterial and fungal communities of 34 matched healthy individuals. Initially, 40 samples were collected, but 6 were not included due to either insufficient fecal sample collection or failed QC. Thus, samples from 34 patients  were included in the final analysis. All participants were from the metropolitan Cleveland, Ohio area. All had metastatic GEP-NENs ; 29 patients were GEP-NETs,  and 5 were GEP-NECs.Candida, Ascomycota, and species belonging to saccharomycetes, (p = 0.0013), compared to controls , (Bacteroides fragilis (p = 0.031), Eggerthella lenta (p = 0.00028), and Bacteroidetes Prevotella (p = 0.032) species compared to lower grade (G1 & G2) GEP-NETs , A. In addGEP-NETs B.Eggerthella lenta, Eubacterium dolichum, and Ruminococcus bromii ; anaerobic, Gram-negative Bacteroides uniformis (p = 0.04); and certain fungi (Trichosporon asahii), (p = 0.005) compared to non-smokers  and certain bacterial species such as the Gram-positive non-spore-forming bacilli  compared to those who deny alcohol consumption  had significantly more fungal species such as  compared to patients who did not receive antibiotics. Patients who ingest probiotics regularly had more abundance of certain bacterial , and fungal species .Additionally, we analyzed the microbiome profile of GEP-NEN patients who regularly consumed probiotics or had taken antibiotics in the 6 months before sample collection. The results indicated that patients who took antibiotics in the last 6 months had relatively more abundance of certain fungal species  which were different from those who consumed mainly an animal protein based diet . Patients with a high carbohydrate diet and those who consumed sweets, on average, six times per week had higher bacterial species, such as Bifidobacterium Adolescentis, Blautia producta, and Agrobacterium  [We compared the microbiome profile according to the dietary habits to identify the relationship between habitual dietary intake and microbiome variation. Patients who consumed vegetables at least five times a week had certain bacterial and fungal species (ctively) ,15 , colorectal carcinoma (CRC), and head and neck carcinoma (HNCC) [Candida albicans can produce cytolytic toxic peptides called candidalysin and acetaldehyde, which are known to disrupt the epithelial barrier function and increase intracellular reactive oxygen species (ROS) that lead to mitochondrial dysfunction and cytoxicity [We found significant differences in the bacterial (bacteriome) and fungal (mycobiome) species between patients with GEP-NENs and healthy subjects, with an increase in the relative abundance of fungi, especially subjects . Certaina (HNCC) . There atoxicity ,24. ThusBacteroides fragilis (B. fragilis) species. Previous studies have shown that B. fragilis colonization in the intestinal tract and production of B. fragilis toxin (BFT) can disrupt the intestinal environment and lead to chronic inflammation and tissue injury, which has correlated with tumorigenesis including colorectal cancer [B. fragilis and the BFT gene were found to be significantly higher in colorectal cancer patients compared to healthy controls [BFT exposure in the human colon may induce chronic IL-17–dependent inflammation, causing epithelial barrier damage and oxidative DNA damage, resulting in increased risk of colorectal carcinoma [B. Fragilis and advanced colorectal cancer stages given that the presence of BFT gene in patients with stage III cancer was significantly higher than in patients with stages I and II cancers [BFT was detected particularly in the late stages of colorectal carcinoma patients [B. fragilis with high-grade NETs and NECs need further exploration.Our results indicate that there are significant bacteriome differences between histological subgroups of GEP-NENs. Patients with high-grade, well differentiated GEP-NETs and poorly differentiated GEP-NECs had significantly enriched l cancer ,26. B. fcontrols . It has arcinoma ,26,27. T cancers . These rpatients ,29. TherEnterobacter hormaechei and T. asahii) compared to those with GEP-NETs. Several previous studies have been performed to determine the interaction between Enterobacter species and cancer. Dilşad Yurdakul et al. investigated the impact of Enterobacter protein on colon cancer cell lines [T. asahii species are non-Candida fungi found widely in nature, and are related to superficial and opportunistic invasive infections in cancer patients. Studies have shown that T. asahii is a part of the dysbiosis signature of mycobiota in several cancers [T. asahii, alter the host immune system and correlate with the development and progression of colorectal cancer [Additionally, patients with poorly differentiated GEP-NECs had significantly enriched bacteria and fungi  GEP-NETs were also associated with significantly higher n cancer ,34. The Bacteroides fragilis toxin and Colibactin expressed by Escherichia coli can cause indirect DNA damage, and are associated with colorectal carcinogenesis [Helicobacter Pylori (HP) infection that has been shown to activate the PI3K-AKT-mTOR pathway, which plays a relevant role in the pathogenesis and progression of gastric NENs [Previous studies have highlighted the role of the microbiome in cancers, however there is no specific cause–effect relationship that has been conclusively demonstrated. Several mechanisms have been proposed concerning how dysbiosis induces carcinogenesis. These include chronic inflammation triggering cell transformation, stimulation of pro-inflammatory cytokines , alteration of the tumor microenvironment, and production of reactive oxygen species (ROS) and nitric oxygen synthase (NOS) that can lead to DNA damage ,36. For ogenesis ,38. The ric NENs . Interesric NENs . TherefoOur study has some limitations including small sample size, and its retrospective nature. Other limitations include the lack of investigating the impact of therapies on microbiome for enrolled patients; furthermore, there was a lack of uniformity with regards to the therapy received by patients. Despite these limitations, this is the first retrospective study investigating the role of microbiome in patients with different GEP-NENs differentiation and grades.We provide the first analysis of the bacteriome and mycobiome in patients with GEP-NENs. The results show significant differences between histological subgroups of GEP-NENs. Further studies are needed to validate these results in larger numbers of patients and to determine the impact of the various hormones they produce, as well as the effect of the various therapies, including somatostatin analogues, on the bacteriome and mycobiome. These studies should concentrate on the potential role of bacteria, fungi, and viruses in the development and progression of GEP-NENs and how they may impact therapeutic responses. Understanding the relationships between different microbiome species and GEP-NEN tumorigenesis will shed light on the pathogenicity of these organisms and may lead to the discovery of novel therapeutic approaches, which could have relevant clinical implications in modifying therapy of these tumors.


New heteromaterials,particularly those involving nanoscale elementssuch as nanotubes, have opened a wide window for the next generationof materials and devices. Here, we perform density functional theory(DFT) simulations combined with a Green’s function (GF) scatteringapproach to investigate the electronic transport properties of defectiveheteronanotube junctions (hNTJs) made of  carbon nanotubes (CNT)with a boron nitride nanotube (BNNT) as scatterer. We used the sculpturenemethod to form different heteronanotube junctions with various typesof defects in the boron nitride part. Our results show that the defectsand the curvature induced by them have a nontrivial impact on thetransport properties and, interestingly, lead to an increase of theconductance of the heteronanotube junctions compared to the free-defectjunction. We also show that narrowing the BNNTs region leads to alarge decrease of the conductance, an effect that is opposite to thatof the defects. Carbon nanotubes have different electronic behaviors;they can be semiconductors or conductors depending on the chirality,which is often difficult to control in synthesis.14 Boron nitride nanotubes (BNNTs), on the other hand, which were firstdiscovered in 1995,15 exhibit a wide energygap of approximately of 5 eV.16 A variety of techniquescan be used for manufacturing CNTs and BNNTs, such as arc discharge,19 laser ablation,22 and chemical vapor deposition(CVD),25 which usually produce again a mixture of chiralities.Various research works have been undertaken on BNNT, which mainlyfocused on properties such as the electronic structure,27 and optical,30 thermal,32 and mechanical33 properties. Regarding the electronic structure, introducing defectsin nanotubes has a nontrivial influence on their electronic structure,34 e.g., defect-induced deformations in CNTs38 lead to an extreme decrease in the electronic transport, whereasin BNNTs, defects41 produce a narrow energy gap. The most common defectsin nanotubes are five–seven  defects.45 Previous studies including molecular dynamics (MD) calculations50 reported that such defects are stable even at high temperatures.On the other hand, modulating the periodic structure of pristine CNTsby other materials such as nitrogen and boron to form heteronanotubes,can modify their electronic properties,54 turning, for instance, a metallicCNT into a semiconductor.57Since theirdiscovery by SumioIijima in 1991,63 CNTs. Heterojunctions, such as carbon–boron nitride heteronanotubejunctions are also energetically stable65 and have a conductance that can be tuned by alteringthe width of the boron nitride strips in the heteronanotube.66 Despite many difficulties involved in the preparationof carbon–boron nitride heterojunctions, experimental effortshave successfully synthesized them using different methods.72 In this work, we investigate the electronic transport propertiesof ideal heterojunctions consisting of  CNTs as left/right leadsand a  BNNT as scattering region. We also consider the impactof different factors such as defects and the width of the scatterer(BNNT) on the transport properties.A variety of experimental techniques have confirmed the synthesisand characterization of nitrogen/boron-doped64 which providesa methodology for creating deterministic carbon nanotubes (CNTs) throughthe reconstruction of bilayered graphene nanoribbons (bi-GNRs), includingbilayered heteronanoribbons materials, such as graphene (G) and boronnitride (BN). Notice that, even though the strain can be large andact as a destabilizing factor in the initial stages of the formationof the nanotubes, the structures naturally evolve to such configurationsafter several relaxation steps, provided the initial ribbons are closeenough to each other. This procedure opens a wide window of optionsfor constructing topologically unique nanostructures. In this work,we started with AA-staked zigzag bilayered heteronanoribbons (bi-hNRs),each layer composed of graphene-boron nitride-graphene (G-BN-G), toform hNTJs, which consist of  CNTs as left/right leads and aboron nitride nanotube (BNNT) as scatterer (CNT-BNNT-CNT). To optimizethe initial supercells (AA-staked zigzag bi-hNRs), we employed theSIESTA73 implementation of density functionaltheory (DFT). We used the local density approximation (LDA)74 with the Ceperley–Alder (CA) exchangecorrelation functional, and double-ζ polarized (DZP) basis setsof pseudoatomic orbitals, together with norm-conserving pseudopotentials.Notice that the LDA usually overestimates the binding, giving distancesbetween atoms a bit smaller than the real ones, but works properlyin general to describe van der Waals interactions75 such as those present in the initial stages of the designof the sculpturenes. The initial supercells were relaxed until allthe forces were smaller than 0.005 eV/Å. To avoid the interactionbetween neighboring heterostructures, a vacuum space of 50 Åwas included along the X and Y directions,whereas along the Z direction the structures wereperiodic. For the leads calculations, a k-point gridof 1 × 1 × 30 in the Brillouin zone was used. Once the finalhNTJs were achieved, we took the mean field Hamiltonian (MFH) andoverlap matrices from SIESTA, and, using the GOLLUM76 implementation of equilibrium transport theory, we calculatedthe low bias electron transmission coefficients T(E) and currents I.To form the heteronanotubejunctions (hNTJs) shown in 1,T1*, T2, and T2*.In an ideal case, the upper/lower edges in the boron nitride regionwould have the T1 and T1* configurations  schematicallyshows the defects created in the relaxed BNNT (scatterer) of the hNTJsdisplayed in Figure S1, we can seethat a variety of defects with different geometries arise in the resultinghNTJs  when the sculpturenesare made. Junct-2 and Junct-3 are relaxed with one defected side (bottom),while Junct-4 and Junct-5 are relaxed with a top/bottom defected side. Figure S1. From this table, we can see that the total number defects in Junct-2,Junct-3, Junct-4, and Junct-5 are 7, 15, 19, and 30, respectively. We conveniently referfrom now on to the hNTJs shown in T(E) of the hNTJs shown in T(E) fromthe left lead to the right lead in the absence of the extended molecule CNT), which, according to the Landauer description,is equal to the number of open channels (NOC), since these structures(leads) are periodic in the Z direction.55 From the same figure we can see that the T(E) of the hNTJs is obviously inhibited due to the presenceof the BNNT. Notice also that Junc-1 is not periodic, since it hasthe BNNT scatterer, but it is free of defects. However, contrary towhat one would expect, the defected hNTJs show in general  higher conductances thanthe defect-free Junct-1. This result, which it is a somewhat surprisingbehavior that ensues from the fact that the defect-free system ishighly insulating, can be explained by the increase of electron delocalizationgenerated by the emergence of dangling bonds at the defects (see below).In what follows, we investigatethe transmission coefficient 77 as we see in the BNNT regionof Junct-1  and heptagon (seven-atom ring), lead to bend the nanotubes withdifferent angles, and also tune the electric properties of the nanotubes.86 Note that sharp bends in CNTs and BNNTs have been observed.89 BNNTs,90 which are perfect insulators,might be turned to semiconductors (reduce the energy gap) by bendingthe tubular structure. Having a look at Figure S2 also show that Junct-2 and Junct-4have the highest delocalized states around the curvatures and defects,which further confirms the previous claims.From  Junct-1 2a, whereI) of the hNTJs, shown in 91qe = |qe| is the electron charge, h is Planck’sconstant, T(E) is the electronictransmission coefficient calculated with GOLLUM, f is Fermi–Dirac distribution function, f(E – μ) = 1/(1 + eE−μ)/KBT, with μLL and μRL being the electrochemical potentials ofthe left lead and right lead, respectively, and T is the temperature. The resulting currents are shown in To provide anotherevidence of the transport enhancement, we calculatethe maximum current  and currents of Junct-6 and Junct-7. As can be seen,there is an extreme drop of the conductance compared to the conductanceof the hNTJs shown in EF, from −0.1eV to +0.1 eV, as shown in Figure S2. Fromthis figure it is clear that the defects in Junct-2 and Junct-4 giverise to more delocalized states around the EF, leading to possible paths through which the electrons cantravel more easily, whereas the defects in the rest of the heterojunctions do not give additionalstates in the BN part around EF, keepingthe central part roughly insulating. This explains then the enhancementin conductance and current of Junct-2 and Junct-4.For a deeper understanding of the evolution ofthe conductanceand current of the heterojunctions shown in EF in T(E)should lead to enhancement of the Seebeck coefficient (S) of the hNTJs. To demonstrate this, we calculate S for all hNTJs, shown in Figure S3. Theobtained values of S, listed in Table S1, are remarkable and illustrate again that hNTJs incontact with ideal CNTs can lead to a rather good thermoelectric performance.More research would be needed in any case to further demonstrate theenhancement in the thermoelectric properties of the hNTJs, which isnot the main target of this work.Additionally,we note that resonances that appear around Conclusions: In this work, we have investigatedthe electronic and transport properties of defects that might be createdin hNTJs using the sculpturene method, or manufactured by other methods,and the influence of other factors such as the curvature induced bythe defects. Our results show that the transmission from left leadto right lead through the defective BNNTs is significantly reducedcompared with that for ideal CNTs. On the other hand, the presenceof defects and the induced curvature lead to an enhancement in theconductance of hNTJs, compared to that of the free-defect hNTJ. Wealso demonstrated that narrowing the BNNT region leads to remarkabledecreases of the conductance. Our LDOS analysis explains the hightransmission of Junct-2 and Junct-4, which show highly delocalizedstates around EF, while for the otherhNTJs, shows no evidence of such states in the scattering region.Furthermore, our findings demonstrate that defects give rise to improvementsin the Seebeck coefficient (S) of the hNTJs. This work lays then thefoundation for the design of nanoscale sensors that, through the changein their transport properties, can act as detectors of particles thatgenerate defects in materials.


The otorhinolaryngological and neurological exams were within normal limits, except for unilateral left hearing loss . Magnetic resonance imaging showed a tumor in the internal acoustic canal, an image suggesting a vestibular schwannoma (VS). Surgery done through a middle fossa approach revealed a tumor that was redder and more adhered than usual, measuring about 10×5mm. It was located atypically in the internal acoustic canal – between the nerves – rather than in the classical posterior position.Facial paralysis House-Brackmann grade IV presented postoperatively.Histology reported a tumor compatible with meningothelial meningioma, which was confirmed by immunohistochemistry . Audiometry was done 15 days later, demonstrating that hearing was preserved (PTA – 62.5dB). The patient recovered from facial paralysis, which had decreased to grade II six months postoperatively.,,Differentiating MEs from VSs may be difficult when MEs are exclusively IC. Both tumors affect similar age groups (45–55 yrs) and predominate in females. They also present with similar signs and symptoms, such as hearing loss and tinnitus.,ICME surgery has certain peculiarities. Compared to ICVSs, ICMEs tend to adhere more and to be more vascularized; they may also occupy various portions of the internal acoustic canal.Immunohistochemistry is useful in differentiating these tumors. MEs may express both epithelial and mesenchymal markers, reflecting their double embryological origin or mesenchymal cell totipotentiality. Many markers have been used, although there is wide variation of results in the literature, which may be credited to differences in methodology.


Symphalangism is a rare, congenital syndrome involving ankylosis of the interphalangeal joints. We present a rare case of fracture at the level of a fused proximal interphalangeal joint in a patient with proximal symphalangism of the hand. Nonoperative management with splinting resulted in osseous healing and restored baseline function. Symphalangism, described by Dr. Harvey Cushing in 1916, is a rare, hereditary syndrome involving the ankylosis of the interphalangeal (IP) joints of the fingers and/or toes . The proMany physicians may be unfamiliar with this condition and have difficulty interpreting its radiographic findings, especially given the various degree of ankylosis. Distinguishing the radiographic findings of symphalangism from degenerative changes or occult trauma may be challenging. Limited literature exists on the management of fractures associated with symphalangism. We present a rare case of fracture through a completely fused PIP joint in a patient with congenital proximal symphalangism of the hand.Our goal is to increase hand surgeons’ and radiologists’ familiarity with this rare congenital condition and present our experience with nonoperative management of a fracture complicating this condition. We also review the available literature, including the underlying genetics and resultant biomechanics of this condition. Our hypothesis is that PIP fusion increases the mechanical lever arm of the applied force, potentially predisposing to fracture and adversely affecting subsequent healing.A 39-year-old, right-hand dominant Caucasian male was referred to hand clinic for evaluation of sequelae of an injury to his left ring finger, which he sustained 5 months prior to presentation. He was working as an automobile mechanic, when he ‘jammed’ his ring finger. He did not immediately seek medical attention, believing it to be a sprain. Over the next several weeks, he had persistent discomfort at baseline and pain while performing vigorous, manual labor. He initially presented to his rheumatologist four months after the initial injury. Radiographs revealed a cortical irregularity at the level of a congenitally fused left ring finger PIP joint. The patient was prescribed celecoxib and referred to hand clinic for further evaluation and management. He was a non-smoker.The patient reported chronic stiffness of his fingers since childhood but was able to adjust and successfully perform manual labor as an automobile mechanic without significant disability. He had previously undergone a negative rheumatology workup for polyarthralgia. Multiple members of his family, which belonged to the Old Order Amish, had ‘stiffness’ and early-onset osteoarthritis spanning several generations.Evaluation of the patient’s hands revealed that the PIP joints of his bilateral index through small fingers postured in extension with associated absence of corresponding dorsal skin creases. Bilateral small finger distal interphalangeal (DIP) level camptoclinodactyly was also noted. There was no active or passive flexion of the bilateral index through small finger PIP joints. Mild swelling and tenderness were present at the left ring finger PIP joint. He had moderate pain with attempted passive flexion of the joint, but no gross instability. The metacarpophalangeal (MCP) and DIP joints of the affected digits exhibited above average active and passive flexion. The remaining non-ankylosed joints were grossly normal in appearance and demonstrated no obvious degenerative changes. The patient had satisfactory overall function of his bilateral hands. He was able to strongly grasp objects with good compensation at the MCP and DIP levels. He was neurovascularly intact. Examination of lower extremities also revealed PIP level ankylosis of his toes bilaterally and mild pes planus. The patient demonstrated no signs of hearing loss.Radiographs of his bilateral hands revealed complete bony ankylosis in extension of the PIP joints in his bilateral second through fourth digits . The DIPClinical and radiographic evaluation were consistent with subacute fracture/delayed union complicating underlying bony proximal symphalangism (Grade III) (as opposed to incomplete fusion). The patient was treated with a custom thermoplastic volar finger-based splint immobilizing the PIP ‘joint’ in full extension, allowing motion at the MP and DIP levels. Discontinuation of the celecoxib was recommended. Radiographs at 3-weeks follow-up revealed persistent cortical irregularity but less prominent lucency and soft tissue swelling at the PIP level. At 6-weeks follow-up, the patient was pain-free. His ring finger was completely nontender and clinically stable with manipulation at the PIP level. Radiographs revealed mature trabecular bridging across the fracture with restoration of the congenital bony fusion . The splSymphalangism is typically inherited in an autosomal dominant pattern. It has been classified into 3 types: (1) true symphalangism , (2) symbrachydactylism (involving digits that are short and stiff), and (3) symphalangism with associated anomalies  ,10.Symphalangism may additionally be categorized into 3 grades, based on the degree of fusion  [Baek 8]Symphalangism is often associated with other skeletal abnormalities such as camptodactyly, clinodactyly, syndactyly, radiohumeral fusion, pes planus, bilateral hip dislocation, tarsal coalition, and congenital fusion of the cervical or thoracic spine . The fusRecent literature continues to elucidate the underlying genetic causes of proximal (PIP level) symphalangism (SYM1). SYM1 is typically an autosomal dominant condition most commonly associated with mutations in the Noggin (NOG) or Growth differentiation factor 5 (GDF5) genes . Noggin Management of congenital symphalangism often consists of benign observation, as patients typically adjust well to the limitations of this condition. In the pediatric population, occupational therapy (OT) may improve dexterity and even increase mobility in cases of symphalangism with residual motion. Surgical treatment of Grade I or II symphalangism has been reported, including joint release, dorsal Z-plasty and/or interposition arthroplasties ,12,20. Tin situ achieved osseous union at 6-weeks despite delayed presentation. He was able to successfully return to his prior manual job and exhibited no complications or sequelae of his injury at 1-year follow-up.PIP joint symphalangism, with its various degrees of ankylosis, may potentially complicate interpretation of hand radiographs. Low-grade (non-bony) fusions may be misinterpreted as simple arthritic changes. Conversely, subtle secondary fractures may be hard to distinguish from higher grade fusions. While congenital symphalangism has been well described in the literature, we are aware of only been two prior reports of symphalangism complicated by fracture. Both failed conservative management. The first case was a 28-year-old gardener that presented with a 2-month history of index finger pain after suffering a direct blow. Radiographs revealed distal symphalangism of the index, middle, and ring fingers with a fracture through the DIP joint of the index. Immobilization in a stack splint for 8 weeks was unsuccessful in achieving union. Surgical intervention consisting of removal of residual cartilage, bone grafting, and K-wire fixation for 6 weeks was successful in achieving osseous union . The secσ = (Mc)/I, where σ is the bending stress, M is the bending moment, c is distance from the neutral axis, and I is the moment of inertia around the neutral axis [It has long been established that other ankylosing disorders of the spine and hip predispose to fracture. The ankylosis creates a longer lever arm and therefore a larger moment . This laral axis . These lral axis ,28. In tral axis . Our patWe present a 39-year-old male with proximal (PIP) symphalangism complicated by fracture. Conservative management with splinting achieved bony healing at 6 weeks and excellent outcome, despite delayed presentation. Knowledge of the spectrum of severity and appreciation of the increased biomechanical stress may allow the hand surgeon to appropriately recognize the atypical findings on radiographs and properly manage this rare condition.Our institution does NOT require ethical approval for reporting individual cases or case series.There is NO information  in the submitted manuscript that can be used to identify patients.


It is paramount to assess the psychometric properties of self-assessment tools in order to check the tests' reliability and validity, also to enable proper outcome interpretation.to check the psychometric properties of the IOI-HA  in its Portuguese version, called QI-AASI , in terms of internal uniformity, correlation between the items and reproducibility. Study design: descriptive, observational and cross-sectional.the questionnaire was deployed to 53 hearing aid users, 34 females and 19 males, with ages between 19 and 92 years - from incomplete basic education to complete higher education, encompassing subjects with monoaural and binaural sound amplification.the QI-AASI had a Cronbach Alpha of 0.69. In the correlation among the items, there were numerous significant correlations. The instrument was properly reproducible, except for item # 6, which presented a significant difference in comparing test and the retest.the QI-AASI is suggested in the rehabilitation process of users of hearing aides; nonetheless, the questionnaire can be difficult for subjects with low social and economic status when self-employed. Hearing is one of the most important senses, as it is the basis for language acquisition and development. In today's world, communication is a fundamental aspect in the insertion of individuals into social life; communication disorders may thus introduce a series of burdens in the life of individuals suffering from them.Hearing aids are sound amplification devices used to facilitate communication involving people with hearing loss. A well-fitted device improves the user's quality-of-life, bringing comfort, a sense of well-being and minimizing the barriers posed by hearing loss.Various questionnaires have been developed with the purpose of assessing the benefits yielded by hearing aids in daily life situations; among them is the IOI-HA . This questionnaire has been translated into 21 languages, and in Portuguese it is known as the QI-AASI . This assessment tool is made up by seven questions used to subjectively look into the results yielded by the hearing aids under the following parameters: 1 - time for which hearing aids have been used; 2 - benefit; 3 - residual limitation in daily life activities; 4 - satisfaction; 5 - residual restrictions to participation; 6- impact on other people; 7 - quality of life. Five graded responses can be given to each question, ranging from poor performance (1) to best performance (5)Tools aimed at measuring the degree of benefit perceived by hearing aid users must be reliable. Therefore, to analyze the psychometric properties of the IOI-HA is of paramount importance in order to test how reliable and valid the intended measurements are and for its results to be accurately interpreted. The psychometric parameters of the IOI-HA have been described for other languagesTherefore, this papers aims to analyze the psychometric properties of the Portuguese version of the IOI-HA.This study is connected to a research project called 'Reproducibility of questionnaires in Portuguese for validating hearing aid fitting' approved by the Ethics Committee a tour institution under permit 2006-347H.The questionnaire was applied to hearing aid users based on the following enrollment criteria: unilateral or bilateral hearing aid users; subjects had to be 18 or older; absence of perceptible cognitive disorders; able to respond the questionnaire without the help of third parties; not having undergone speech and hearing therapy and/or changes to the hearing aid between testing and retesting.Seventy-two subjects were called based on information locally available and through referrals. Two of the subjects refused to participate on the study. After they were informed on the purpose of the study, a visit was scheduled for them to respond to the questionnaire. Seventeen other individuals were excluded from the study as they failed to meet the above mentioned requirements, which left us with a final sample of 53.On the first visit, all subjects agreed to participate on the study and signed an informed consent term to then fill out a questionnaire containing personal identification data, general information on the type of hearing loss and on the use of hearing aids. They were then given the IOI-HA. The subjects answered the questions by themselves, without the help of third parties, as required by the researcher. Instructions were included in the text preceding each question, and subjects could pick only one answer for each question.Lesser educated participants and subjects with reading difficulties were read the questionnaire, with questions and possible answers being presented to them in the very same form as they were written. These individuals were grouped separately. In cases of doubt over any of the given questions, the researcher either told the subjects to read the question again or read it to them.After the questionnaire was applied for the first time, a new appointment was scheduled for retesting, within at least seven days to no more than thirty days. Participants responded the questionnaire again following the same procedure described above, and were not given access to the answers they gave the first time around.A total of 53 hearing aid users made up the studied sample; 34 (64.2%) were females and 19 (35.8%) were males, with ages ranging between 19 and 92 years. All subjects lived in the State of Rio Grande do Sul, southern Brazil. In terms of formal education, 1.9% of the subjects reported not to have concluded basic schooling; 13.2% concluded basic schooling; 9.4% did not conclude fundamental education; 20.8% concluded fundamental education; 1.9% dropped out of high school; 28.3% concluded high school; 3.8% did not conclude higher education programs; and 20.7% concluded higher education programs.Binaural use of hearing aids accounted for 24.5% of the sample, while monaural use amounted to 75.5%. Hearing aid types ranged from analog to digital devices, programmable and non-programmable ones. The time for which hearing aids were used by the subjects ranged from 2 months to 32 years; 71.7% of the subjects had been using hearing aids for under 10 years.The sample was divided into two groups, one of 48 subjects who answered the questions without anyone's help and another with 5 in which the researcher aided them by reading the questions and possible answers.The answers given to the questions by the subjects were converted into numeric values to allow for statistical analysis. Calculation was done based on the answers given the first time the test was applied. Another two individuals had to be dropped from some of the statistical analysis, as their questionnaires had not been completely answered; we were thus left with a sample of 46 for some of the questions.Mean values and standard deviations were then analyzed for the entire questionnaire and each individual question. The correlation between each item and the whole test, i.e., the correlation between each item and the total score on the questionnaire was also calculated using Pearson's Correlation Ratio. According to the literatureThe Cronbach Alpha is one of the procedures required to estimate the reliability of a test; there are no rules establishing reference values for ratios, but values above 0.75 are deemed highFactors are groups of questions covering different areas, i.e., each factor comprehends a group of questions connected to each other. As also done for the questionnaires in English, Dutch, and GermanThe results obtained from testing-retesting were analyzed and compared using the paired T-test with a level of significance set at 5%. The correlation between pairs was verified again using Pearson's Correlation Ratio with a level of significance set at 5%.The correlation between the items in the questionnaire is presented on The IOI-HA factorial analysis resulted in the extraction of three factors. Factor 1 covered items 2, 3, 4, and 7, connected to benefit, residual limitations to everyday-life activities, satisfaction, and quality of life. Factor 2 was made up by items 5 and 6 on residual restriction to participation, and impacto f hearing loss upon others. Factor was represented only by item 1, on the time for which subjects had been using hearing aids.Finally, This study looked into the psychometric properties of the Portuguese version of the IOI-HA in terms of internal consistency, correlation between items and reproducibility.The mean value for each item, as shown in The mean values of the questions for the group of subjects who had the questions read to them ranged between 3.80 and 4.80, a finding quite close to the values seen in the 46-subject sample, indicating that merely having the questions read to may not impact the outcome of the questionnaire.The questionnaire's internal consistency was measured through the Cronbach Alpha ratio, and a value of 0.69 was found as seen on In the correlation between items presented on The factors extracted from the QI-AASI are different from the ones mentioned in the literatureIn this study the items were grouped in a different manner, as seen in the results. Given that the studied populations are different, it is perfectly possible that questions are grouped differently. In this case, Factor 1 covered items referring to the benefits or limitations that hearing aids produce upon the users. Factor 2 was made up by items related to users and the environment. And Factor 3 was constituted only by the question on time of use of hearing aids unrelated to the other two factors.The differences between the mean values in testing and retesting are presented on This study presents some preliminary results from the analysis done on the psychometric properties of the Portuguese version of the IOI-HA. We could see that the questionnaire presents moderate levels of internal consistency. Many items are correlated to each other , and the tool can be reproduced adequately, except for item 6 (impact on others), which had statistically significant differences when comparing testing and retesting values.As also seen in other studiesAt last, further studies should be conducted to verify the psychometric properties of the IOI-HA using more extensive samples and other test application contexts.