Avian influenza virus subtype H9N2 is identified in chickens with respiratory disease while Bacillus cereus (B. cereus) has been frequently isolated from chicken feed in China. However, the roles of co-infection with these two pathogens remain unclear. In the present study, SPF chicks were intragastrically administered with 108 CFU/mL of B. cereus for 7 days and then inoculated intranasally with 100 EID50 of H9N2 three days later. Alternatively, chickens were initially inoculated with H9N2 and then with B. cereus for one week. Post administration, typical respiratory distress persisted for 5 days in both co-infection groups. Gizzard erosions developed in the groups B. cereus/H9N2 and B. cereus group on 7th day while in group H9N2/B. cereus on 14th day. More importantly, both air-sac lesions and lung damage increased significantly in the co-infection group. Significant inflammatory changes were observed in the B. cereus group from day 7 to day 21. Moreover, higher loads of H9N2 virus were found in the co-infected groups than in the H9N2 group. Newcastle Disease Virus (NDV) specific antibodies were decreased significantly in the H9N2/B. cereus group compared to the B. cereus and the B. cereus/H9N2 groups. Nonspecific IgA titers were reduced significantly in the B. cereus group and the H9N2/B. cereus group compared to the control group. In addition to this, lower lymphocyte proliferation was found in the con-infection groups and the H9N2 group. Hence, feed-borne B. cereus contamination potentially exacerbates gizzard ulceration and aggravates H9N2-induced respiratory distress by inhibiting antibody-mediated immunity and pathogen clearance. Thus controlling the B. cereus contamination in poultry feed is immediately needed. Bacillus cereus (B. cereus) is among the microorganisms most often isolated from cases of food spoilage and gastrointestinal diseases as well as non-gastrointestinal infections due to its ability to produce several enterotoxins such as the heat-stable emetic toxin i.e., cereulide and tissue-destructive enzymes1. This opportunistic pathogen leads to vomiting and diarrhea syndromes in livestock and human beings, which is associated with rapidly fatal clinical infections, especially in neonates and immunocompromised individuals. Among cumulative food poisoning infections, as a cause it stands at third important position after Salmonella species and Staphylococcus aureus, and approximately from 102 to 104CFU/g has been reported in some food2. In a survey, contamination with 1.3 × 108 CFU of B. cereus contributed to egg deterioration which was largely eliminated at low temperature3. A recent report indicated that B. cereus isolation rates in litter material, droppings, birds feed, liquid manure and raw milk was found to be 93.3%, 78.9%, 41.2%, 100.0% and 9.8%, respectively in local 10 dairy farms. Milk-borne B. cereus might represent a potential hazard to consumers due to inactivated during milk manufacturing4. On the contrary, improved immune status of piglets by B. cereus var. toyoi has been reported which could be by inducing a higher CD4+/CD8+ ratio and improving the functions of systemic immune cells5. B. cereus BC7 could efficiently detoxify zearalenone (ZEN) both in vitro and in vivo, indicating a potential feed additive for removing ZEN in a mouse model6.B. cereus can survive, thus absconding this hostile environment. Escaping of B. cereus from macrophages could be by hijacking an active cellular process or causing the lysis of the cells, remains unknown, perhaps could be a cytotoxic factor in action7. Moreover, genetic background of B. cereus is nearly alike that of B. anthracis allowing it to escape the immune system8.Additionally, within the macrophages spores of Ornithobacterium rhinotracheale, Aspergillus fumigatus, and Chlamydia psittaci leads to unadorned pneumonia and increased mortality in SPF birds, threating to poultry health and leading to a huge economic loss9. A G57-genotype H9N2 emerged due to antigenic variation and facilitated adaptability in chickens. H9N2 was predominant in farms of vaccinated chickens in China, leading to outbreaks in 2010–201310. In our pioneer study, both H9N2 virus and B. cereus have been frequently isolated from the lungs of birds showing respiratory distress while up to 80% of feed has been found contaminated with B. cereus (Unpublished). However, the pathogenic mechanism of any disease associated with B. cereus and H9N2 is unclear. In the present study, our hypothesis is that immune suppression by B. cereus causes aggravation of the respiratory distress after avian influenza subtype H9N2 infection. The experiments in our study were designed to evaluate both humoral and mucosal responses post inoculation with the combinations of B. cereus and H9N2 or H9N2 alone.In current China poultry industry, co-infection of avian influenza H9N2 subtype virus with B. cereus C isolates was basis on biochemical tests and PCR assay. The DNA extracted produced the expected 1500 bp, 759 bp, 935 bp, 618 bp, 635 bp and 565 bp PCR products, respectively from 16SrDNA gene, nheA gene, nheB gene, nheC gene, Em1gene and CytK gene .Identification of ene Fig. . A sequeB. cereus, B. cereus/H9N2 and H9N2 alone. When compared to the breathing difficulty of birds in the H9N2 group on day 3, the breathing difficulty in birds inoculated with H9N2/B. cereus con-infection persisted for one week. By day 5, typical diarrhea was observed in groups B. cereus alone, B. cereus/H9N2 and H9N2/B. cereus. No birds died in any group during this study.By day 3 post inoculation (PI), the infected chickens showed respiratory distress with open-mouth breathing and severe anorexia in groups H9N2/P < 0.05) decreased in group H9N2/B. cereus in comparison with groups H9N2 or B. cereus. By day 14 PI, weight gain decreased significantly (P < 0.05) in groups H9N2/B. cereus and B. cereus/H9N2 as compared to that of groups H9N2 or B. cereus. By day 21 PI, weight gain differed non-significantly among all groups  on day 7 PI  in the B. cereus/H9N2  group as compared to the lesions present in other groups  greater in the two co-infection groups as compared to the control group on days 14 and 21  burden of the H9N2 virus than in either of the two co-infected groups on day 14. However, a lower (P < 0.05) H9N2 virus burden was still evident in the B. cereus/H9N2 group than that of the H9N2/B. cereus group. While non-statistical difference in viral burden among the three groups on day 21 was found  increase of the B. cereus load was recorded in the H9N2/B. cereus group compared to other two groups on day 14 while on day 21, the B. cereus load was non-significant difference among the three groups  and B. cereus (P < 0.01) in comparison with  those of the B. cereus/H9N2 group at three-time points. A statistically significant decline in NDV-specific antibody levels was consistently noted in the H9N2/B. cereus group as compared to the B. cereus group from day 7 to day 21 (P < 0.05)   Fig. . IgA ant01) Fig. .Figure 6B. cereus and H9N2 group had a significantly (P < 0.05) decreased lymphocyte proliferation index than in the birds of control or B. cereus/H9N2 group  decreased in IL-2 expression in comparison to that of the controlgroup or H9N2 group. Moreover, IL-2 expression was reduced significantly (P < 0.05) in the B. cereus group as compared to that of the control group. Whereas, non-significant difference between two co-infected groups was found  in both the B. cereus/H9N2 and the H9N2/B. cereus groups compared to the B. cereus or H9N2 group on day 14 , and haemorrhagic inflammation in the lungs. A primary infection with H9N2 followed by inoculation with B. cereus caused birds to develop more severe breathing difficulty in comparison with H9N2 infection alone. More interestingly, higher lesion scores in the air-sacs and lungs were diagnosed in groups B. cereus/H9N2 and H9N2/B. cereus while H9N2 virus concentrations were significantly higher in the groups B. cereus/H9N2 and H9N2/B. cereus than in the H9N2 group on day 14. The above facts warrant our hypothesis that primary contagion with avian influenza virus subtype H9N2 followed by B. cereus inoculation aggravates avian respiratory distress.In the present study, birds orally administered 12, resident macrophages13, and recruited white blood cells due to the direct virus cytopathic effects14. The performed experimentations in our study were planned to identify the pathogenesis of B. cereus under such conditions. In current study, chickens received orally 10 × 8 CFU/ml of B. cereus for 7 consecutive days, which was comparable to the administration of the fast-growing broiler per day. The high inoculation is based on 100 g feed consumption per SPF chicken on day 21. In our preliminary survey, average colonies of B. cereus were determined to be 1 × 106 CFU per gram in the feed. Main cause of complications and mortality following an avian influenza virus attack, are the secondary bacterial infections. Combined infection with influenza A virus from human infection  and Bacillus thuringiensis (107 spores) from a commercial source leading to 100% mortality in mice indicates that non-pathogenic Bacillus species will pose a high risk during bird flu outbreak15.During seasonal outbreaks of avian influenza, secondary bacterial infections are favored by the changes in respiratory tract epitheliumB. cereus or AIV H9N2 alone, but a severe fibrinous exudate, typical pneumonia, GEU were detected in the groups B. cereus/H9N2, H9N2/B. cereus and B. cereus alone. This suggests that B. cereus administration produced significant gizzard erosions and lung injury, while co-existence with H9N2 aggravated such respiratory disease. The present data showed that the most severe lung lesions were observed in the groups B. cereus/H9N2, H9N2/B. cereus and B. cereus, indicating that hemorrhagic inflammations is induced by B. cereus contamination in the poultry industry.In the current study, no chickens died when inoculated with the co-infection of AIV H9N2 with Bacillus cereus infection results in gizzard erosions and lung lesions, it might contribute to damage to the digestive tract and respiratory epithelial cells, facilitating infection by other pathogens. The mucosal immune system must balance tolerogenic responses against respiratory tract commensal microbiota, while preserving the ability to support protective inflammatory responses against attacking pathogens. This delicate balance is maintained through the harmonized signals and interactions of a variety of specialized cells16. Besides providing an important barrier between invading pathogens and the underlying tissue, lung epithelial cells can interact with bacteria via both macrophages and antibacterial peptides. Once the respiratory mucosal barrier is damaged, infection by other pathogens will be facilitated17. In the present study, non-specific IgA levels were decreased significantly in the B. cereus group and H9N2/B. cereus group at two-time points, suggesting mucosal immune suppression. IgA protects the mucosal surface against bacterial destruction and lower IgA levels would be expected to facilitate development of gizzard ulceration and hemorrhagic pneumonia in the B. cereus group and H9N2/B. cereus group. Also, low levels of IgA might favour B.cereus and H9N2 survival in the lungs of chickens.As B. cereus group on days 7, 14 and 21. Consequently, low IgG-specific antibody leads to vaccine failure and NDV outbreak in poultry flock. This might be associated with numerous NDV cases in broilers post immunization with live attenuated vaccine. Both air-sac lesions and lung damage were found significantly higher in the birds in the groups B. cereus/H9N2 and H9N2/B. cereus as compared to the control group. Aforementioned findings suggest that B. cereus might facilitate H9N2 infection, leading to vaccine failure. The present results indicate that both B. cereus/H9N2 and H9N2/B. cereus inoculation protocols induced lower lymphocyte proliferations on day 14. The fact that birds in the co-infection groups exhibited severe air-sac lesions and lung injury suggests a poor innate immune response against infection of H9N2 in the lungs. In general, host defence and Th1 responses are more effective against intracellular pathogens (viruses and bacteria that are inside host cells)18. Regarding cytokine secretions, co-infection of B. cereus and H9N2 induced lower IL-2 and IFN-γ while IL-6 levels were upregulated in contrary to those of the control group, indicating imbalance in the immune response of Th1/Th2 and the reduced IL-2 in the groups initially infected with B. cereus might facilitate AIV H9N2 to survive and more severe lesions in the lungs due to Th1/Th2 imbalance. The high concentration of IL-2 is associated with CD8+ T cells expansion and differentiation in conforming to the antiviral immune response, and the reduced IL-2 concentration might lower the capacity to control the virus load19. This might account for the obstinate avian airsacculitis and failure of vaccination plans in the poultry industry. However, no significant difference of IL-12 was found among all the groups from day 7 to day 14. Post administration with IL-12 in mammals, NK cells were stimulated to produce IFN-γ through their constitutive mien of IL-12R20 and production of IFN-γ by IL-12 was in a dose-dependent manner21. In this study, no significant difference of IFN-γ was found among the B. cereus group, H9N2 group or combination of two pathogens due to IL-12 secretion.Secondly, the dysfunction of antibody-mediated immunity is associated with poor development of NDV-specific antibodies, characterized as the lower IgG response that was found in the H9N2/B. cereus might pose a risk for human health. H9N2 influenza virus is not limited to birds and frequent cases of humans suffering with H9N2 virus infections are on the recorded22. H9N2 influenza virus is distinct from H5 and H7 influenza viruses, some of these are exceptionally pathogenic, could be due to the multiple basic amino acids presence in the hemagglutinin (HA) cleavage site as defined by the Office International des Épizooties (OIE). All of the isolated H9N2 strains have low pathogenicity in accordance with the OIE cataloging. In spite of this fact, H9N2 virus infections have caused serious disease in several cases, even high mortality in some cases in domestic poultry have been reported23. On the other hand, consumers have easy access to food-borne B. cereus pathogens via contamination, particularly the biotype of B. cereus infection, which would expect to aggravate H9N2 respiratory diseases by suppressing the host immune response, potentially leading to a human pandemic during a seasonal influenza outbreak. This is the first study to reveal that B. cereus aggravates H9N2 infection in chickens. Further studies should check whether B. cereus-mediated lung inflammation contributes to respiratory diseases with other pathogens, generation of H9N2 mutants and vaccination failure in the poultry industry.Importantly, food-borne B. cereus, as a primary or an enduring latent infection can cause lung inflammation in vivo, and may increase other-pathogens susceptibility, such as H9N2. Our findings suggest that H9N2/B. cereus infection contributes to NDV vaccine failure and severe respiratory distress by eliciting a damaged cellular immunity.In conclusion, 25. Briefly, the birds were monitored 2 or 3 times daily for clinical symptoms and chickens displaying clinical signs were euthanized. Birds were euthanized with an overdose of CO2 using a gradual fill (30% chamber volume/min) device. The CO2 movement was continued for a minimum of 1 min after the loss of respiratory signs. When death was confirmed, an additional secondary physical euthanasia  was carried out before sampling and carcass discarding.Five 10-day-old SPF embryonated eggs and 105 SPF birds of 21 days age were purchased from Weitong Merial Laboratory Animal Co., Ltd, Beijing and kept at the Experimental Center for Animals, China Agricultural University (CAU), Beijing, China. All birds were kept in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Council of the People’s Republic of China. The experimental protocols were approved by the Committee for Experimental Animal Management of CAU and followed humane protocols to minimize animal pain as described previouslyB. cereus C type were then carried out employing 16SrRNA gene, nheA gene, nheB gene, nheC gene, Em1gene and CytK gene. Additionally, the 16SrRNA gene sequence was analyzed by a commercial institute . H9N2 AIV/chicken/Shandong/2011 was isolated from broilers as described formerly21. Due to the potential for aerosol infection of avian influenza A H9N2 for human, respiratory protection was required for all activities conducted at Biosafety Level 2 (BSL-2).Lungs were collected aseptically from the diseased chickens. Streak cultures were performed using Mannitol Yolk Polymyxin (MYP)  and incubated at 37 °C under aerobic conditions for 24 h. The colony of interest was identified by Gram stain and biochemical assays. Biochemical characteristics of the bacterial culture, as well as molecular identification of B. cereus group birds were inoculated intragastrically with 1 mL (1 × 108 CFUs/mL) of the liquid culture of B. cereus each day for 7 consecutive days. H9N2 group birds were inoculated intranasally (it) with 0.2 mL 100 EID50 H9N2 virus. B. cereus/H9N2 group birds received intragastrically with 1 × 108 CFUs of B. cereus for 7 days and then 100 EID50 H9N2 virus in 0.2 ml via the intranasal route H9N2/B. cereus group birds were administered intranasally with 100 EID50 of H9N2 virus, and then administered 1 × 108 CFUs/ml of B. cereus intragastrically for one week. Control group birds were intranasally treated with the same amount of sterile physiological saline. All animals were given SPF feed free of B. cereus. Clinical signs/symptoms, body weight, and mortality were recorded a minimum of twice daily and the monitoring period continued till the end of the experiment.One hundred and five SPF chickens were randomly divided into 5 groups with 21 chickens per group  samples were examined as previously reported26. Briefly, 6 chickens were euthanized at days 7 and 21 post-infection and the lungs were lavaged three times with 1.0 mL sterile saline. Suspensions were centrifuged at 1000 rpm for 5 min and stored at −80 °C until used. The concentration of IgA of the BALF was measured using an ELISA kit . The concentrations of IL-2, IL-6, IL-12 and IFN-γ from 6 BALF samples per group were determined using commercial kits .Seven blood samples per group were collected from jugular vein punctures at days 7, 14 and 21 post-infection (PI), and NDV-specific antibodies were measured using a commercial kit . In addition to weight gains, 8 peripheral blood lymphocyte proliferations were determined on day 7 and day 14 using a BrdU cell proliferation ELISA kit 27 . For B. cereus determination, the samples from the lungs were inoculated onto an MYP agar plate (containing 50 μg/g of Colistin sulfate B) and incubated for 24 h at 37 °C. Finally, the number of bacterial colonies was measured.The lungs were aseptically removed on days 14 and 21, respectively. Briefly, sterile lung tissues of 7 birds from each group were taken and then minced, 50 mg lung tissues from each bird and supernatants from lung homogenates were obtained. The homogenates were stored at 4 °C for 40 min and then centrifuged at 2000 rpm/min for 5 min. The supernatants were divided into two aliquots and maintained at −80 °C until used. One stock was used to determine the concentration of H9N2 virus by inoculating into five 10-day-old SPF embryonated eggs and the eggs were examined for 48 h. Thereafter, HI titer of allantoic fluid from each egg was tested to determine the 50% egg infective dose (EIDP < 0.05.Data are expressed as mean ± SEM (standard error of the mean) and analyzed using STATISTCA v.7 (Stat Soft) software. Nonparametric analysis and Mann–Whitney U tests were performed for comparison between groups and the data presented as median values. Multiple group analysis, including the multiple comparison correction (Bonferroni) was carried out. Statistically significant differences were judged as All experimental protocols were approved by the Committee on the Ethics of Animal Experiments of China Agricultural University (Permit Number: 20151110–160). This study does not involve the use of human sample or tissue.Supplementary information


This study investigates long-term changes in the variability of TC intensity of global tropical cyclones, a topic which has been relatively infrequently studied to date. Our study finds that the variability of global TC lifetime maximum intensity (LMI), as measured by the LMI standard deviation, increases during 1981–2016. The increasing trend in LMI variability is statistically significant for both the Northern and Southern Hemispheres, with three individual TC basins: the western North Pacific, the South Indian and the South Pacific also having statistically significant increases. This increasing trend primarily results from distinct changes in the relative percentages of TCs with different intensities. When comparing two periods: 1981–1998 and 1999–2016, the proportions of weak and strong TCs increase, whereas moderate TCs occur relatively less frequently. This bimodal pattern of observed LMI distribution change is further linked to opposite trends in the average intensities of TCs that undergo rapid intensification (RI) during their lifetime (RI TCs) and those that do not (non-RI TCs). The LMI distributions of RI and non-RI TCs migrate to higher and lower intensities, respectively. Our results demonstrate from an observational perspective that strong TCs have strengthened while weak TCs have weakened as the global climate has warmed since 1981. A majority of modelling studies estimate a decrease in the total frequency of global TCs accompanied by an increase in the average number of intense TCs in response to anthropogenic global warming5. This anticipates a future increasing tendency in the average intensity of global TCs, which is consistent with significant increasing trends in both the mean lifetime maximum intensity (LMI) of TCs and the intensity of the strongest TCs derived from historical satellite-based temporally homogenized datasets. By contrast, a few publications project increasing TC frequency13. Due to the lack of theoretical understanding of storm occurrence8, there is greater inconsistency in projections of future TC frequency than intensity. It also remains uncertain how global warming affects changes in LMI variability for global TCs as well as individual basin TCs. This uncertainty can influence both the verification of TC simulations in climate models and the prediction of TC activities in different intensity categories.The influence of climate change on tropical cyclone (TC) activity has generated considerable scientific attention during the past ~20 years14. There exists a bimodal pattern of storm LMI PDFs over the globe as well as for individual TC basins17. Two peaks in the PDF are separately related to LMI distributions of TCs that undergo rapid intensification (RI) during their lifetime (RI TCs) and TCs that do not (non-RI TCs)17. In several modeling studies, both the overall LMI distribution and the peak of the LMI PDF are skewed to higher intensities in a warmer climate, which induces an increasing and decreasing proportion of strong and weak TCs, respectively26. This change could significantly modulate the LMI mean but also influence the LMI variance. In contrast, a few climate models represent a more complex change in the LMI PDF under high-CO2 conditions28. Although the majority of the LMI PDFs are shifted to higher intensities in warmer scenarios, the relative frequency of weak TCs also increased in some simulations28. However, this feature was rarely discussed in the literature, perhaps because there exists a great deal of disagreement as to the definition of weak TCs in climate model outputs. Since TCs in climate models are usually defined by objective tracking algorithms, the identified storm activity is highly sensitive to the specific thresholds in tracking techniques from different modeling studies which utilize different models and may have different model resolutions30. Compared with strong TCs, it is harder to correctly identify weak TCs using various tracking methods in climate models, which results from the challenges involved in separating weak TCs from disturbances and waves. Due to these uncertainties, modeled activity of weak TCs is not discussed as extensively as that of strong TCs in most previous studies. Moreover, compared with modeling studies, there have been few publications on the observed changes of the PDF of LMI. Many modeling papers utilize the observed LMI PDF to verify their historical simulations and then project future changes in the simulated LMI PDF (e.g. ref.13).LMI variability is primarily determined by the probability density function (PDF) of LMI, serving as a fundamental feature of the TC climatology31 and the Joint Typhoon Warning Center (JTWC)32 as archived in the International Best Track Archive for Climate Stewardship (IBTrACS)33 v03r10. We use data during the satellite era (1981–2016) to investigate the potential long-term trend in TC LMI variability, which is measured by the standard deviation (STD) of the annual LMI sample. We find that there exist significant increasing trends in the annual TC LMI STD over the globe as well as for some individual basins. These trends are primarily caused by differences in the LMI PDF between the two sub-periods (1999–2016 minus 1981–1998), which represents a bimodal pattern with two peaks occurring at higher and lower intensities. Furthermore, the different trends in annual averaged intensities of RI TCs and non-RI TCs are responsible for the bimodality of the distribution of the LMI PDF change. Here RI is defined as an increase of at least 30 kt in TC intensity within a period of 24 h or less35.In this study, we utilize TC LMI records from the National Hurricane Center (NHC)−1 .When the annual STD of LMI is computed from the IBTrACS data during 1981–2016, a significant upward trend is represented over the globe, with an increasing rate of 0.22 kt yr−1 Fig. . There a−1 Fig. , with th−1 that is about twice the LMI STD trend  is applied here to provide descriptive statistics for skewed distributions as the LMI PDF. There are significant increasing trends of LMI IQR from 1981 to 2016 for the globe, with a rate of 0.48 kt yrend Fig. . Similarend Fig. , while t36), whereas there is great uncertainty in the trends related to weak TCs in climate projections. Bimodal patterns are also seen in the LMI PDF changes over the NH and SH, although the peaks of the distributions are somewhat different between the hemispheres  distribution corresponding to a larger  STD. The increasing LMI STD is primarily caused by the LMI PDF becoming flatter, which is shown as the LMI PDF difference between 1981–1998 and 1999–2016 Fig. . This meres Fig. . Moreove17. When the annual LMI averages in the period of 1981–2016 are calculated for different TC groups  storms, which usually exhibit stronger (weaker) intensities, have become increasingly stronger (weaker) during the past two decades. These observed trends are in agreement with ref.42. In general, TCs generated at lower latitudes can reach greater peak intensities due to a longer time spent in a conducive environment , whereas TCs forming at higher latitudes typically do not achieve as high intensities due to reduced sea surface temperature and enhanced vertical wind shear. Here we take the WP which exhibits the most significant poleward shift of storm tracks and examine trends in the annual averages of WP TC genesis location for RI and non-RI storms 31 and the Joint Typhoon Warming Center (JTWC)32 . For comparison, we also utilize data from the World Meteorological Organization (WMO)-sanctioned forecast agencies for basins outside of the North Atlantic and the eastern North Pacific, which are listed as IBTrACS-WMO.TC best-track data are available at 45. The percentages of global TCs with a duration less than 24 h and 48 h are 5.7% and 12.4% during 1981–2016, respectively. Our results are not significantly impacted when excluding TCs with a lifetime shorter than 24 h and 48 h, respectively.The LMI is defined as the peak one-minute maximum sustained wind achieved by a TC during its lifetime. Only storms with an LMI greater than 34 kt are considered in our study in order to minimize possible influences of the temporal evolution of observational technologies for tropical depressionst-tests are applied to calculate significance levels. Total global TC numbers average ~85 TCs per year, while those for the Northern Hemisphere and South Hemisphere average around 60 and 25 TCs, respectively.The trends of time series are obtained using linear least-squares. Two-tailed Student The LMI PDF is defined as the percentage distribution of storm LMI in the nearest 5-kt bins. The LMI PDF change refers to the difference between the LMI PDFs in 1981–1998 and 1999–2016.Supplementary Information


Mucormycosis following hematopoietic stem cell transplant (HSCT) carries a very high mortality rate. Pulmonary mucormycosis often leads to systemic dissemination and eventual death. It is imperative for transplant providers to have a high level of suspicion for mucormycosis and initiate early treatment. Here, we present a 64-year-old woman who died of disseminated mucormycosis 13 days following her allogeneic HSCT.  A 64-year-old female with a history of acute myeloid leukemia (AML) presented for allogeneic HSCT and passed away from intracerebral hemorrhage secondary to mucormycosis infection 13 days following her transplant. On autopsy, it was found she had angioinvasive mucormycosis in her frontal lobe leading to cerebral edema which eventually led to tonsillar herniation and brainstem infarction. Her lungs were the likely source of infectious dissemination.  This case represents an unusual course of events following HSCT in that no other published case shows tonsillar herniation resulting from mucormycosis-related intracerebral swelling. We also report this case because it is believed mucormycosis in HSCT patients is underreported. Additionally, our case highlights the importance of increased vigilance for mucormycosis in patients with prolonged neutropenia prior to HSCT and the potential link of voriconazole prophylaxis and increased risk for mucormycosis. Infections after hematopoietic stem cell transplant (HSCT) are of particular concern due to their high morbidity and mortality. HSCT places patients at a considerable risk of infection, depending on the severity of immunodeficiency, pathogen exposure, and time since HSCT .Mucorales [β-D-glucan test and Aspergillus galactomannan test also do not detect components of the mucoralean cell wall. Therefore, most times the diagnosis can only be made with direct tissue sampling, which in the case of mucormycosis often occurs post-mortem [One infection with a particularly high mortality is mucormycosis , a term used to describe fungal infections caused by filamentous fungi belonging to the order ucorales . On a daucorales . Pulmonaucorales . CT scanucorales . Howevert-mortem .When undiagnosed, pulmonary mucormycosis invades the pulmonary vasculature then spreads to the heart, mediastinum, and blood. Around 12% of mucormycosis cases occurs after a bone marrow transplant . MortaliThe above information makes it imperative for providers taking care of HSCT patients to have a high level of suspicion for mucormycosis and initiate early treatment. Here, we present a particularly devastating case to highlight its relatively indolent course until invasion of a vital organ, in this case the brain, occurs. Additionally, we report this case because it is believed mucormycosis in HSCT patients are likely underestimated due to low rates of autopsy  and becaA 64-year-old woman with a history of refractory acute myeloid leukemia (AML), hypertension, and left ear deafness was admitted for allogeneic bone marrow transplant and subsequently died of intracerebral hemorrhage secondary to mucormycosis 13 days after receiving her stem cells.This patient was originally diagnosed with AML 8 months prior to HSCT because of fatigue and pancytopenia on lab work. Peripheral smear at the time showed 43% blasts, and a subsequent bone marrow biopsy showed 60–70% myeloblasts consistent with a diagnosis of AML. Genetic studies done of the bone marrow showed deletions of 16q and 5q, and FLT3 was negative. She was started on induction treatment with 7 + 3 cytarabine/idarubicin which was complicated by neutropenic fever and AML retinopathy.Neutropenic fever occurred on day 2 of induction treatment admission for which she was given piperacillin/tazobactam until she became afebrile on day 13 of admission. Fevers recurred on day 15 of admission for which cefepime and metronidazole were started. CT scan of her chest was done on day 16, showing a new right lower lobe opacity concerning for pneumonia along with 9 nodules in both the lungs, all less than 6 mm. Treatment dose voriconazole was added for fungal coverage. On day 19, she was broadened to meropenem and anidulafungin due to recurrent fevers. A repeat CT scan of her chest including her sinuses was done showing resolution of her previous consolidation with no evidence of significant sinus disease and no change in her lung nodules. She became afebrile on day 4 on meropenem and anidulafungin but was continued on these antibiotics until day 47 of admission awaiting her absolute neutrophil count (ANC) to reach 500. She was then switched to oral ciprofloxacin, amoxicillin/clavulanic acid, and voriconazole. She completed a total of 7 days of oral antibiotics and then was put on prophylactic voriconazole and acyclovir until HSCT.After induction, she suffered from prolonged neutropenia and never reached an ANC above 1,130 in the time between the first induction and her death . Her treatment after 7 + 3 consisted of cladribine, mitoxantrone, decitabine, and venetoclax with venetoclax finally achieving remission. At the time of transplant, her disease status was in complete remission with incomplete hematologic recovery (CRi) due to sustained cytopenia. On the last office visit prior to being admitted for HSCT, she was noted to have no symptoms and felt overall well.In terms of further imaging after initial diagnosis, she had a repeat CT scan 7 days prior to HSCT as part of her pretransplant workup to monitor the nodules previously seen. This scan showed stable nodules, all of which did not change in size or appearance. No consolidation or new findings were seen.The patient was admitted and started on trimethoprim-sulfamethoxazole, fluconazole (switched to voriconazole one day prior to HSCT), and valacyclovir prophylaxis. She then started receiving a myeloablative conditioning (MAC) regimen with total body irradiation (TBI)  divided into 6 fractions). Two days following TBI completion, she received her donor leukocyte infusion (DLI), followed by cyclophosphamide and mesna 3 days after DLI. She then received her CD34+ stem cells 6 days following DLI infusion. She had her first recorded fever of 101.0 on day (−5) of her transplant, one day following DLI infusion. From this day until the date of death, she was noted to have daily fevers, as high as 104.0. Blood cultures were drawn daily, all of which showed no growth. Sputum cultures were not obtained due to lack of productive cough, and urine cultures showed no growth. She was initially started on cefepime for neutropenic fever which was eventually broadened to meropenem then to piperacillin-tazobactam and vancomycin. She was also switched from prophylactic voriconazole to treatment dose voriconazole 2 days prior to death. Switching to amphotericin B or brain imaging was not considered upon first fevers due to lack of consideration for mucormycosis.Twelve days after receiving her stem cells, she started complaining of severe right shoulder pain; a chest X-ray was obtained which showed a right upper lobe consolidation. CT scan of the chest was done which showed ground glass consolidation with interlobular septal thickening and mucoid impaction. The day after the shoulder pain started, the patient became increasingly fatigue and more lethargic but was answering questions appropriately. Later that afternoon, she was noted by the nursing staff to be completely unresponsive. The patient was emergently intubated and taken for a noncontrast CT scan of the head which showed extensive cerebellar edema with peripheral hemorrhage in the cerebellar folia and petrosal surface of the cerebellum with upward herniation. There was also marked compression of the brainstem and effacement of the fourth ventricle with lucency of the brainstem compatible with ischemia  and assoMucor spp. as can be seen in On autopsy of the brain, it was noted the patient had duret hemorrhage in the pons, cerebellar intraparenchymal hemorrhages and associated subarachnoid hemorrhage with cerebellar edema, right frontal lobe hemorrhagic infarct with angioinvasive fungi, and a small left thalamic hemorrhage. On fungal stain of the right frontal lobe, it was noted she had Enterococcus and very light growth of Serratia marcescens. Fungal cultures showed light growth of Mucor spp. AFB stains were negative. Fungal staining showed large, nonseptate hyphae with 90 degree angle branching and nonparallel walls consistent with Mucor spp. Again, no PCR or FISH testing was done due to identifiable appearance on microscopic examination. Examination of the sinuses showed no significant disease.Her gross exam of her right lung showed a firm dark-red mass on the right upper lung lobe, measuring 9.5 × 8.0 × 6.8 cm, and the cut surfaces of this mass reveals a well circumscribed, homogenous, round, dark-red fleshy mass resembling a hemorrhagic infarction. On microscopic examination, extensive hemorrhagic areas, pulmonary edema, and small pulmonary infarct areas around some vessels occluded with thrombi were found. Aerobic and anaerobic cultures showed very light growth of vancomycin-resistant The sequence of events most likely leading to her death was concluded to be frontal lobe fungal invasion causing edema and hemorrhage leading to cerebellar tonsillar herniation, brainstem hemorrhage, and ultimately death.Mucor spp., this was the likely reservoir for disease dissemination.The above case represents a fatal course of mucormycosis following HSCT. Likely, it was our patient's prolonged neutropenia prior to HSCT which put her at a much higher risk for mucormycosis, after obvious colonization. The MAC regimen further suppressed her immune system to a point where dissemination to other organs systems outside of the lung was inevitable. In our search of the literature, only case reports exist of mucormycosis leading to intracerebral or intracerebellar hemorrhage. Takahashi et al.  report aOne aspect of our case which did not necessarily fit the typical course for mucormycosis after HSCT is the timing of infection. In a study conducted by Xhaard et al., they found infection occurred at a median of 225 days after allogeneic HSCT but with a wide range of 0–2693 days . In a stThe above type of mucormycosis is referred to as disseminated mucormycosis and carries a very high mortality . CurrentUnfortunately, in our patient, antibiotics were not changed to AmB or an antifungal agent with activity against mucormycosis. Ultimately, given the severity of overall fungal burden, it is unclear whether initiating appropriate antibiotic therapy at pulmonary symptom onset would have halted the progression of disease. Rather, it is imperative to identify patients who have prolonged neutropenia prior to HSCT as being at higher risk for mucormycosis. These patients should be put on posaconazole 200 mg three times daily prophylaxis as per ESMID/EFISG/ECMM guidelines . Additionally, their guidelines state that if mucormycosis is a potential differential diagnosis, biopsy should be pursued  . BecauseMucorales, but the clinical implications of these findings remain unknown [Another important point about our case is our patient's long-term voriconazole prophylaxis prior to HSCT. In a review of voriconazole-associated zygomycosis, it was noted that voriconazole prophylaxis has been associated with increased numbers of mucormycosis. Additionally, they state that voriconazole exposure can modulate the virulence of some  unknown . Our patThe combination of prolonged neutropenia, pulmonary nodules, and long-term voriconazole prophylaxis should have prompted further workup of mucormycosis prior to HSCT. We report this case as a reminder for other practitioners working with HSCT patients to be vigilant of the risk of mucormycosis and to be diagnostically aggressive in order to prevent the outcome we present here.


Sensorineural hearing loss is caused by the loss of sensory hair cells (HCs) or a damaged afferent nerve pathway to the auditory cortex. The most common option for the treatment of sensorineural hearing loss is hearing rehabilitation using hearing devices. Various kinds of hearing devices are available but, despite recent advancements, their perceived sound quality does not mimic that of the “naïve” cochlea. Damage to crucial cochlear structures is mostly irreversible and results in permanent hearing loss. Cochlear HC regeneration has long been an important goal in the field of hearing research. However, it remains challenging because, thus far, no medical treatment has successfully regenerated cochlear HCs. Recent advances in genetic modulation and developmental techniques have led to novel approaches to generating HCs or protecting against HC loss, to preserve hearing. In this review, we present and review the current status of two different approaches to restoring or protecting hearing, gene therapy, including the newly introduced CRISPR/Cas9 genome editing, and stem cell therapy, and suggest the future direction. Hearing loss can be divided into sensorineural and conductive hearing loss. Conductive hearing loss is a biophysical problem, resulting from the fixation or disruption of the ossicular chain, middle ear effusion, and third window of the cochlea. In most patients these problems can be surgically managed. By contrast, sensorineural hearing loss is caused by the loss of sensory hair cells (HCs) or damage involving the afferent nerve pathway to the auditory cortex. These types of damage are caused by a variety of ototoxic agents, such as aminoglycoside and cisplatin, acoustic overexposure, and mutations in the genes responsible for hearing and aging. They are mostly irreversible and result in permanent hearing loss.The current clinical option for sensorineural hearing loss is hearing rehabilitation with hearing devices, which range from externally worn to implantable devices. Yet, despite recent advances in hearing aid and cochlear implant technologies, the perceived sound quality does not mimic that of the “naïve” cochlea. Impaired speech perception in noisy environments and musical sound perception are well-known drawbacks of cochlear implantation , 2 and rHC regeneration is one of the most important goals in the field of hearing research. In the past two decades, differences in HC characteristics among species and between sensory organs have been explored. Unlike mammalian HCs, the HCs of avian species  regeneraBecause exposure to ototoxic and acoustic insults is sometimes unavoidable, protecting HCs from possible ototoxic insult has also been considered, and drugs able to prevent hearing loss related to various ototoxic insults have been studied but, thus far, without clinical success –10, one In this review, we introduce two different approaches to restoring or protecting hearing. The first is gene therapy Figures , in whichttps://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm574058.htm).The history of gene therapy began in the 1960s and early 1970s, when genetically marked cells were developed and used to understand the mechanism of cellular transformation by several viral vectors. With the introduction of, and advances in, recombinant DNA techniques and gene cloning, cell-based trials demonstrated the possibilities of repairing defective genes in vitro. The development of retroviral vectors and other gene transduction methods allowed for more efficient phenotype corrections in animal models . FollowiNevertheless, in the treatment of hearing loss, gene therapy must overcome several obstacles related to the characteristics and anatomy of the structures of the inner ear. The cochlea is a closed, fluid-filled cavity covered by bone, and it is very vulnerable to changes in the amount and composition of inner ear fluid. Consequently, the delivery of therapeutic materials into the cochlea without damaging homeostasis is very challenging. A possible route is the round window, which is the only membranous structure covering the cochlea and the exit site of the wave traveling from the oval window (point of connection to the ossicles). Another option is to insert material inside the cochlear cavity to create an opening, in a procedure called a cochleostomy. This was the approach used by our group to inject material into the three cochlear cavities , because via the round window only the scala tympani is accessible.Viral vectors can be used to deliver a gene or small molecule able to modulate gene expression into target cells in the cochlea. The viral vectors most commonly used for this purpose are adenovirus (Adv) and adenoassociated virus (AAV). Other candidates such as retrovirus and lentivirus were also used but were relatively less safe and effective . For use in vitro and ex vivo using RNAi [ in vivo studies. The areas of research using viral vector gene therapy or RNAi can be divided into genetic hearing loss, hearing protection, and HC regeneration. Recently, more efficient targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has been introduced and will be discussed in detail in the following sections.The discovery of RNA interference (RNAi) which is a natural process of knocking down the targeted gene presents an alternative method to modulate gene expression . There aing RNAi –29. Desping RNAi , 31, notThe screening, diagnosis, and rehabilitation of genetic congenital hearing loss have become well established, to the benefit of patients and, in affected children, their parents. With the aid of a hearing aid and cochlear implant, patients with genetic congenital hearing loss can receive proper education and care. However, as noted above, currently available devices do not completely replicate the quality of sound received by the naïve ear, and the use of the electronic device is cumbersome. Genetic mutations resulting in hearing loss can already be screened for in utero. Ideally, a genetic mutation involving hearing loss that is found before birth would be treated by replacing the defective gene by gene therapy. In utero gene delivery has been achieved in animals and some studies have shown that cure was achieved. Methionine sulfoxide reductase B3 (MsrB3) is an important protein for auditory function in mammals. Its depletion leads to the degeneration of stereociliary bundles and the death of HC, resulting in severe congenital hearing loss. Delivery of the MsrB3 gene using AAV virus directly to the otocyst at E12/5 rescued hearing function after birth .One of the common gene mutations related to human genetic hearing loss involves GJB2, which encodes connexin 26, a gap junction protein that enables the recycling of toxic material such as potassium. The lack of connexin results in the accumulation of potassium in the extracellular fluid and thus a decrease in endocochlear potential, eventually causing hearing damage. Using an animal model of the GJB2 mutation, Yu et al. were able to restore the depleted connexin by gene therapy ; howeverMice with VGLUT3 mutations lack vesicular glutamate transporters and exhibit hearing loss by disrupted synaptic transmission. Studies in humans suggested that missense mutations in the human gene SLC17A8 (encoding VGLUT3) are related to the progressive loss of hearing at high frequencies, as observed in DFNA25 mutations. In an animal model of hearing loss related to a VGLUT3 mutation, histologic and functional recovery of hearing was achieved when gene therapy using AAV was administered before the maturation of hearing function . SimilarThese very promising outcomes  suggest In modern society, ototoxic exposure, for example, to aminoglycosides and cisplatin, as well as acoustic overexposure, is commonplace. Although many pharmacologic agents have been investigated, none were shown to be clinically useful in preventing ototoxicity. The advantage of gene therapy over pharmacological approaches is the prolonged expression of the gene and, therefore, of the therapeutic agent. A serious drawback is the inevitable damage of sensory cells during the surgical process. Recent studies using empty AAV vectors in cochlear HCs demonstrated the potential safety of the procedure but further studies with actual vectors carrying the required gene are still needed.The molecules exhibiting protective effects against aminoglycosides when delivered by gene therapy are neurotrophins –42, blc2In inner ear research, the ultimate goal is HC regeneration. Unlike other epithelial cells, which consistently turn over and are replaced once lost or damaged, the HCs of the mammalian cochlea do not regenerate. Theoretically, there are two ways to force HC regeneration: by inducing either proliferation of the auditory epithelium or the direct transdifferentiation of SCs cell to HCs. Using transgenic mice lacking P27 cells –55 or Rbγ-secretase inhibitor to block notch signaling [Transdifferentiation, in which a cell phenotype is converted from one to another, has been previously described in the basilar papilla of birds , 58, theignaling . A combiignaling . Taken t in vitro mutation repair using homologous recombination [This approach is designed based on the protective mechanism of bacteria against viral infection and it is the most recent and advanced among the three programmable nucleases adapted for genome engineering . With thbination . Howeverbination . HoweverStem cells are widely used in the field of regenerative medicine. These undifferentiated biological cells either differentiate into specialized cells or divide by mitosis to produce more stem cells. Both ESCs and induced pluripotent stem cells can be used in the regeneration of specific cells and tissue, as is required when damage to a tissue or other anatomic structure has occurred and self-regeneration is not possible.The use of stem cells in hearing loss therapy has several prerequisites. First, since HCs are not easy to regenerate in the mature mammalian cochlea, whether they can be produced by stem cells is unclear. Second, due to the hostile environment of the cochlea itself, the high potassium level of the endolymph, and the presence of tight junctions that block insertion of the implanted cells into the organ of Corti , survivaIn the following, we introduce the techniques shown to generate HCs from stem cells and discuss the conditioning methods that can increase the survival of transplanted stem cells in the scala media.There are several published protocols to generate the hair-cell-like cell from pluripotent stem cells –79. In mThe differentiation of HCs from embryonic stem cells takes place in three different phases . During Although the differentiation of stem cells to HCs seems to be well established, based on literature reports, a careful step by step reevaluation of the differentiation process is needed to allow its optimization and to minimize potential complications when the method is clinically applied. There are efforts to reduce the steps and to increase the efficiency for the differentiation process by gene modulation , 77. DesGiven the above described information, very hostile environment of the endolymph , including the high potassium concentration and tight junction barrier, the surHere we would like to discuss more about the former option and introduce ways to design less hostile cochlea environment. There are several ways to reduce the high potassium content. One is simply flushing out the scala media fluid and replacing its content with one more hospitable to stem cells, such as stem cell media. Another is the administration of a systemic potassium-lowering drug, such as loop diuretics.These two techniques were used in HeLa cells , which aAlthough these results might be applicable for animals in acute model, there is a possibility that these processes might not be necessary in chronic condition which is more similar to the clinical hearing loss circumstances. It is not obvious whether high potassium concentration will remain after the loss of the crucial structures. Further clarification of ionic composition of scala media after the long duration deafness might minimize the necessity of conditioning procedure.Many new scientific developments can be expected when the in vitro differentiation of stem cells into HCs is combined with in vivo alterations that lead to an environment hospitable for stem cells to survive long enough for their clinical use. Whether fully differentiated HCs can be obtained  in the dAlternatively, we could expand our interest to the cochlear implantees with expected poor outcome due to the degeneration of nervous tissue . With hi trans-splicing, and hybrid [One of the limitations of using AAV, a well described/established recombinant viral vector for gene therapy, is the limited size of genome that could be packaged . The sizd hybrid .Successful animal studies would enable the planning of clinical applications of gene and stem cell therapies in hearing loss patients. However, there are several important issues to consider. These include potential side effects. The viral vectors used in the studies should be tested in detail for their safety, for example, to ensure that they do not change their phenotype by cross-acting with a similar virus and thereby result in a contagious outbreak. As for the genome editing approach, minimizing the off-target effects must be warranted before the actual clinical applications. Stem cells must be properly controlled and optimized to avoid unnecessary cell growth and, thus, a tumorous condition. Another consideration is the socioeconomic effectiveness of gene and stem cell therapies, which, so far, are very expensive due to the high price of stem cells and viral vectors and their genetic modification. Moreover, their effectiveness is still questionable. Finally, the application of these novel approaches clinically to treat hearing loss rests upon the very high probability of hearing restoration.https://clinicaltrials.gov/ct2/show/NCT02132130) using CGF166 which is a recombinant Ad5 serotype containing the human atonal transcription factor (Hath1). Given the recent advances in genetics and cell biology as well as methods of overcoming current obstacles, the ultimate goal in clinical application of novel therapy for hearing loss may not be far away.Despite these limitations, gene and stem cell therapies remain a tempting strategy in hearing loss research because they are the only options that may lead to HC regeneration in the mature mammalian cochlea. Along with the successful outcomes reported in the literature, there is an ongoing human clinical trial of Ad delivered atonal gene (


In summary, this study revealed that the ADL score is lower in this region and identified several influencing factors. These new findings will be useful for the local government to enhance the current LTC program for the elderly population.To investigate the activities of daily living (ADL) and influencing factors, this survey study was conducted in Shangrao of Jiangxi. A total of 1087 elderly subjects in a long-term care (LTC) program participated in this study and their physical function, cognitive ability, self-rated health, and chronic disease were recorded during March 2017 and April 2018. The ADL scale was used to evaluate the health status of the elderly. F-test and multiple linear regression showed that the average ADL of the participants was 15.12 ± 17.59. The incidence of visual and verbal impairment was 68.6% and 14.1%, respectively. Over 74% of the elderly had severe cognitive impairment; and the prevalence of chronic disease was 84.5%. Multivariate analysis revealed that age, education, BMI (Body Mass Index), low income, verbal and cognitive ability, visual status, health self-evaluation, and some chronic diseases were related to self-care ability ( Jiangxi covers 23 districts, 66 counties, and 11 county-level cities including Shangrao city. These cities share similar economic and social conditions. At present, China is launching a long-term care insurance system, which requires individual provinces to select one of its cities as a pilot area to test for this program. Shangrao was selected as the pilot area in Jiangxi for the care insurance in 2017. Our research, which mainly assesses the activities of daily living (ADL) of the elderly who participated in long-term care insurance, is a part of this pilot program.According to the Sixth Population Census in 2010, the number of elderly people aged 60 years and older in China increased to 178 million, reaching 13.26% of the total population [To confront its aging society, China introduced a long-term care insurance system in 2016 . ShangraJiangxi province is located in southeast inland China. The main topography of Jiangxi province consists of hilly and mountainous regions as well as valley and plains. The soil composition is both red and yellow and the land is not fertile. Agricultural production is mainly based on rice farming. Jiangxi is one of the main grain production areas in China. Since it is an agricultural-based province with a low economic foundation, Jiangxi is a relatively underdeveloped region.From March 2017 to March 2018, we surveyed a total of 1087 elderly persons (aged 60 years and above) who received long-term care services in Xinzhou district (23), Guangfeng district (79), Shangrao county (38), Yushan county (48), Hengfeng county (61), Yiyang county (64), Qingshan county (73), Dexing city (114), Wuyuan county (69), Wannian county (75), Yugan county (61), and Poyang county (382). The study subjects covered 12 counties (cities and districts) within the jurisdiction of Shangrao, a city of Jiangxi province. The inclusion criteria included residents who (a) had an official household registration, (b) received long-term care services, and (c) were aged 60 years or above at the time of the survey. The exclusion criteria are as follows: those who refused to participation in investigation and physical examination, and those who lived beyond the areas being investigated. A total of 1109 cases were investigated, but only 1087 participants were enrolled because 22 cases were informed but still rejected. The effective rate of investigation was 98.02%.2)), old-age care type, and type and number of chronic illnesses. The second measure of health is the ADL; the specific score was expressed by utilizing the Barthel Index of Activities of Daily Living [This study used the ADL scale, an internationally accepted measurement of long-term health status . The maiy Living . This scy Living . This scThe survey was completed by door-to-door and face-to-face questions with uniformly trained and qualified investigators. The survey was started in March of 2017 and completed by 27 April 2018. The investigation environment was quiet, and the investigators were responsible for explaining the connotation of each of the investigation projects, while strictly controlling the quality to ensure the accuracy of the data.The main contents of the questionnaire include six different health indicators: the first is basic demographic information, the second is the ADL, the third is physical function, the fourth is the health self-evaluation, the fifth is cognitive ability, which uses the Mini-Mental State Examination (MMSE), and the last measurement is chronic disease. Among these measurements, ADL was measured using the internationally accepted ADL scale, and the specific score was represented by the Barthel Index of Activities of Daily Living. The simple operation intelligence state questionnaire was used for the cognitive ability.p < 0.05 was considered statistically significant.Data were recorded with the Epidata 3.1 database  and SPSS19.0 statistical software  was used for data collation and statistical analysis. The measurement data are mainly described by mean and standard deviation, while the counting data are described by relative logarithm such as rate and composition ratio. The main methods of statistical inference included F-test, while single factors analysis was used to examine the effect of individual independent variables on ADL and multiple linear regression analysis was used to study the effect of multivariate interactions on ADL. Bilateral This study was approved by the Ethics Committee of Jiangxi CDC (Center for Disease Control and Prevention) (Project Identification Code: ZGRSJXFGS015). Informed consent information was attached to each questionnaire and introduced before the surveys.p < 0.001). Furthermore, the higher the education level, the lower the ADL score (p < 0.05). Gender, low insurance and low income, loss of independence, marital status, BMI, and old-age care showed no significant difference in ADL scores among the elderly population studied (p > 0.05)  .n = 341) of participants had normal vision and the rest (746) had a visual impairment. The majority of the responders (934 out of 1087) had normal verbal ability while only 14.1% had a verbal impairment. The ADL score of the visually impaired was 13.12 ± 15.717 and the ADL score of the verbal impaired was 4.67 ± 10.477. A statistically significant difference was observed in the ADL among the following variables: vision and verbal ability (p < 0.001).Among the 1087 elderly subjects aged 60 years or above who participated in long-term care insurance, 31.4% (p < 0.01).The ADL score of the elderly who rated themselves as “good-health” was found to be 22.41 ± 17.881, while 75.7% (823) considered their health status to be poor. The ADL score of the elderly who had normal cognitive ability was 25.71 ± 21.633, as compared to the majority (>74.0%) who had severe cognitive impairment and 19.3% with moderate cognitive impairment. Statistically significant differences were observed in the ADL among the variables of health self-evaluation and cognitive ability (p < 0.001).Of the 1087 elderly people aged 60 years or above who participated in long-term care insurance, 918 had chronic disease, showing a prevalence rate of 84.5% . Among tAnalysis was performed by using the ADL score of the elderly who received long-term care as a dependent variable while age, gender, low income, bereavement, education level, marital status, BMI, old-age modes, type and number of chronic diseases, verbal ability, self-rate health, and vision and cognitive ability were used as independent variables. Multiple linear regression analysis was performed and the methods of assigning independent variables are shown in The health status of the elderly is largely influenced by the economic development of the region . ShangraUsing ADL and MMSE scales, this study revealed that the elderly group had a poor Dalit—an average ADL of only 17.4 ± 17.3 points. By using the MMSE assessment, this study showed that nearly all the elderly (>97%) had some degree of cognitive impairment, with 74.2% exhibiting a severe level of impairment. Compared with the Dabie Mountains of Anhui Province, which has the same economic conditions, this group of elderly people was different from ordinary elderly people in that their ADL and MMSE abilities were very low ,14. CompFindings from this study indicate the need for more care to be provided to the elderly population, increasing the current support and assistance in order to improve the health status of the elderly population including the need for the modification of the current public policies. Their situation fully meets the requirements of China’s current long-term care insurance scheme, and they should be compensated with relevant insurance funds to ensure their daily needs. The new findings obtained from this study would be helpful and important not only for promoting the establishment of an improved standard for long-term care insurance for the elderly population, but also for pointing out the critical improvements needed in the long-term care insurance system in Jiangxi province. In addition, the findings from this study strongly suggest that the pilot program in Shangrao needs to be extended to the entire elderly population in Jiangxi province.The multivariate analysis of the ADL score among the elderly also demonstrated that older age, higher BMI, more chronic illnesses, economic difficulties (low income family), higher education, poor health self-evaluation, lack of language skills, visual loss, and cognitive problems lead to lower ADL scores. These variables are therefore determined to be all factors that affect ADL. These findings are consistent with current notions from many existing literature reports: (1) with the increase of age, the daily activity ability is weakened ,21,22,23Additional factors associated with ADL include financial difficulties , higher education level, lack of language ability, loss of vision, physical health dysfunction, poor self-rated health, and some cognitive problems. There may also be interactions between these factors. For example, a previous report indicated that there is a relationship between aging and a higher prevalence of chronic diseases . In addiIn summary, when a long-term care insurance system and implementation plan is developed, there is a need to fully consider the impact of relevant factors on ADL. In addition to providing a certain amount of care insurance compensation for the elderly, the long-term care plan should also help the elderly to control their weight and reduce their BMI through a series of measures such as health education, health promotion, and behavioral intervention. Furthermore, it is important and necessary for the local government to provide financial aid to those in economic difficulties, to help them overcome difficult situations, to provide physical training programs to help reduce language disability and loss of vision, and to provide personalized health care and health management plan interventions for different chronic illnesses in order to reduce complications. Thereby, the health of the elderly can be maintained, the ability of daily life will be enhanced, and the quality of life will be improved.


As a result, in 1980 WHO developed the International classification of impairments, disabilities and handicaps.International Classification of Functioning, Disability and Health, and accompanied with descriptions of typical problems encountered in daily activities at various levels of severity.The need for an independent classification system for all impairments and disabilities as a complement to the well-established International classification of functioning, disability and health.WHO’s first classification for hearing impairment dates to 1986 and is based on the recommendations of an expert group established by WHO specifically for this purpose; the classification has been modified several times since then. The current one is shown in International classification of functioning, disability and health lowest generic qualifier of mild impairment, but reduced the lower limit from 25 to 20 dB hearing level. This decision was reinforced by the fact that persons with hearing sensitivity < 20 dB rarely benefit from or require amplification devices, and was in agreement with the remarks by the WHO expert group in 1991.In 2008, the GBD Expert Group on Hearing Loss addressed these concerns by reviewing the WHO classification for hearing impairment and other data inputs for the GBD study 2010.International classification of functioning, disability and health specific provisions for hearing problems associated with localization and lateralization, particularly in difficult listening situations. The GBD expert group proposed six categories for bilateral hearing impairment, differentiated consistently by 15-dB steps, since scientific or clinical evidence was lacking to support the variable steps between the grades in the WHO classifications. The choice of 15-dB was intended to reflect the minimum shift in pure-tone audiometry thresholds that is typically considered as clinically and functionally significant, especially in occupational noise surveillance.A separate category for unilateral hearing impairment was introduced in line with the extensive literature on the functional, educational, psychological or social impact of this type of hearing impairment in all age groups.International classification of functioning, disability and health is difficult. However, by definition, a condition is said to be disabling if it causes someone to have an illness, injury or condition that makes it difficult to do the things that other people do. As a result of the expanded concept of disabling hearing impairment, it was no longer critical to have a separate classification for children  would provide more benefit, especially in low-resource settings. The mere recognition of those with mild hearing impairment could be advantageous, especially for school children who may benefit from simple but effective intervention such as preferential seating in the classroom. Such recognition would also help parents and carers to have better understanding of the communication challenges children with mild hearing impairment face in their daily life.However, the term disabling is neither recommended by International classification of functioning, disability and health allows that, wherever possible, the person whose level of functioning is being classified (or the person’s advocate) should have the opportunity to participate and to challenge or confirm the appropriateness of the category being used, and the assessment assigned.Classifications based solely on pure-tone audiometry have limitations.The proposed classification was first reported in the 2010 GBD study to generate prevalence estimates and disability weights for hearing impairment, and has been adopted in all subsequent GBD publications.International classification of functioning, disability and health, especially under the disability-inclusive global agenda of the sustainable development goals.We recommend that WHO considers adopting this revised classification to ensure that future WHO estimates of hearing impairment are aligned with those published periodically in the GBD study and other epidemiological data. Doing so would also ensure that no persons with functional hearing impairment, regardless of the severity, are unduly placed at a disadvantage compared to those with other impairments within the framework of the


Pure-tone screening (PTS) is considered as the gold standard for hearing screening programs in school-age children. Mobile devices, such as mobile phones, have the potential for audiometric testing.This study aimed to demonstrate a new approach to rapidly screen hearing status and provide stratified test values, using a smartphone-based hearing screening app, for each screened ear of school-age children.pass  or fail (possible hearing impairment). The proposed smartphone-based screening employs 20 stratified hearing scales. Thresholds were compared with those of pure-tone average (PTA).This was a prospective cohort study design. The proposed smartphone-based screening method and a standard sound-treated booth with PTS were used to assess 85 school-age children (170 ears). Sound-treated PTS involved applying 4 test tones to each tested ear: 500 Hz at 25 dB and 1000 Hz, 2000 Hz, and 4000 Hz at 20 dB. The results were classified as pass and fail results (pass in 168 ears and fail in 2 ears). The smartphone-based screening detected moderate or worse hearing loss (average PTA>25 dB) accurately. Both the sensitivity and specificity of the smartphone-based screening method were calculated at 100%.A total of 85 subjects (170 ears), including 38 males and 47 females, aged between 11 and 12 years with a mean (SD) of 11 (0.5) years, participated in the trial. Both screening methods produced comparable The results of the proposed smartphone-based self-hearing test demonstrated high concordance with conventional PTS in a sound-treated booth. Our results suggested the potential use of the proposed smartphone-based hearing screening in a school-age population. Worldwide, more than 466 million over 5%) people, including 34 million children, are estimated to have a hearing impairment. Hearing impairment is difficult to monitor because of the limited availability of testing equipment and trained specialists in many developing countries ,2. Unide% people,Pure-tone screening (PTS) is considered as the gold standard for hearing screening programs for school-age children ,12. PTS The Hearing Scale Test (HST) is a novel hearing screening method derived from the consecutive hearing screening procedures for approaching the current hearing status of each screened ear of children ,8. The HAutomated audiometry devices have demonstrated that comparable hearing threshold values, compared with those obtained by automated audiometry, such as computer-assisted audiometry ,10,12 orEar Scale and evaluated its performance and feasibility as a hearing screening program for school-age children. We investigated the accuracy of the hearing tests conducted on mobile devices calibrated by RETSPLs for Apple EarPod [In this study, we developed an iOS-based smartphone hearing test app e EarPod . We compe EarPod ,21-27,29This prospective cohort study was conducted at an elementary school in Taipei, Taiwan. We recruited children from grades 5 and 6, aged between 11 and 12 years. A total of 85 children (38 boys and 47 girls) were enrolled, with 170 ears tested. The trial was approved by the Institutional Review Board of Taipei Veterans General Hospital (2017-10-003CC). Written informed consent was collected by the teachers from the parents, before the scheduled date of the hearing screening tests. After instruction by the researchers, each child, in a random order, underwent smartphone-based and booth-based hearing screening consecutively. The smartphone-based hearing screening procedures were performed in a quiet room in the school. Before the hearing screening, the students were taught how to wear the headphones and push a button when hearing the tone. The air conditioner was turned off during the measurements to reduce ambient noise, the level of which was monitored every 30 min by a sound level meter to ensure an ambient noise level of less than 50 dB at test frequencies of 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz.pass result for an ear indicated that the child responded correctly to all 4 test tones. If the child did not respond to all 4 test tones after 2 consecutive testing procedures, then the ear was assigned a fail result. PTA hearing thresholds of more than 25 dB at 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz in the sound-treated booth were designated as hearing impairment.The audiologist manually controlled a GrasonStadler GSI 18 screening audiometer that was used with a Telephonics TDH-39 supraaural earphones previously calibrated according to International Organization for Standardization (ISO) 389-1. A Ear Scale app (version 2.0) was developed to perform pure-tone air conduction hearing testing and was made freely accessible as a download through the Apple iTunes store in 2018. The HST, a new modified hearing screening method derived from consecutive hearing screening procedures to assess the current hearing status of each screened ear of children, was used to determine the hearing threshold [5; 4 to S1) until the child did not respond to any of the 4 test tones; otherwise, the test stimulus level was increased . Scales S1 to S5 of the HST are equivalent to a PTS pass result, whereas scales S6 to S10 and NR are equivalent to a PTS fail result  values  in each ear was determined by sound-treated booth audiometry. The results from the Ear Scale app were compared with the threshold obtained from sound-treated booth PTA measurement. These data were entered into 2×2 tables to calculate the sensitivity, specificity, positive predictive value, and negative predictive value. The hearing scale obtained from the Ear Scale app and the corresponding mean pure-tone threshold obtained from the sound-treated booth are shown by a box plot . The cor) values .pass and fail results, respectively. Similarly, of the 170 ears tested by the Ear Scale app, 98.8% (168/170) and 1.2% (2/170) of the tests were assigned pass and fail results, respectively  of 13% (11/84), 5 dB (S2) of 38% (32/85), 15 dB (S3) of 33% (28/85), 20 dB (S4) of 11% (9/85), and 25 dB (S5) of 4% (4/85), whereas fail results were stratified as 35 dB (S7) of 1% (1/85). Similarly, 84 pass results and 1 fail result for right ears were also further stratified. The results of 168 pass ears and 2 fail ears are pooled and shown in Of the 170 ears tested by sound-treated booth PTA, 98.8% (168/170) and 1.2% (2/170) were assigned ectively . The resectively . In addiAs the HST was used in our Ear Scale app for the default screening protocol, we also compared the HST with other popular protocols, including those suggested by the AAP and ASHA. The Ear Scale app was highly accurate at the tested frequencies  for all 3 screening protocols. The specificity was 100% and the sensitivity was 100% for HST , 95.2% for AAP , and 95.2% for ASHA . The false-positive rate was 0% in all 3 screening protocols, whereas the false-negative rates were 0% of HST, 4.8% of AAP, and 4.8% of ASHA, respectively. A summary of the results from all 3 tested screening protocols is provided in 1, S2, S3, S4, and S5) in right ears and left ears  to 100 dB (S20), plus an NR result. Our Ear Scale 25 dB (S5) menu item fit a normal hearing range, the Ear Scale 50 dB (S10) menu item fit a mild hearing loss range, the Ear Scale 75 dB (S15) menu item fit a moderate hearing loss range, and the Ear Scale app with the HST from 5 dB (S1) to 100 dB (S20) menu item can be customized for a wide range of hearing loss for school-age children. Conventional PTS provides a pass / fail result, and it therefore provides little information regarding a child’s hearing ability. The Ear Scale app with the HST proposed in this study has 10 stratified hearing scales from 0 dB (S1) to 45 dB (S10) plus an NR result. The Ear Scale app with the HST is derived from the hearing screening concept of dichotomized test results (pass or fail), but the use of computerized hearing screening procedures and hearing scales with different test stimulus levels allows the minimum audible hearing scale to be determined. The scale determined by the Ear Scale app can present the current hearing status of each tested ear. The Ear Scale app with the HST can rapidly evaluate the hearing status of the tested ear, typically within 3 to 5 min.The findings from this study support the use of the Ear Scale app in smartphone-based hearing screening of school children. To the best of our knowledge, this is the first report proposing a method for stratifying hearing test results on a smartphone and then using it for hearing screening in school children. As hearing screening is useful for detecting hearing impairment in the school system , we devepass or fail results (1) to 45 dB (S10) recorded in an initial hearing assessment can be used for further follow-up surveillance in hearing screening programs [5), thus facilitating comparisons of hearing screening results among classes or schools , whereas the false-positive (0%) and false-negative rates (0%) were low when the hearing tests were performed in a quiet room in the school library, ensuring an ideal test for hearing screening. The Ear Scale app was also found to be highly accurate in testing several hearing screening protocols in addition to the built-in HST , includiThe environmental noise level is one of the most common concerns in hearing screening ,30,42,43This paper proposes an innovative approach to hearing screening of school-age children. We developed an Ear Scale app that is comparable with clinical-grade PTS in a sound-treated booth in terms of hearing test results. With favorable high sensitivity and specificity rates and low false-positive and false-negative rates, this study demonstrated that using the proposed Ear Scale app can rapidly screen hearing status and provide stratified test values for each screened ear, and it is therefore an ideal tool for hearing screening in schools.