E. coli (recipient). This novel protocol can be used to measure HGT frequency for both plasmid transgenes and/or genomic transgenes from a donor to recipient organism.Horizontal gene transfer (HGT) is a natural process for an organism to transfer genetic material to another organism that is a completely different species, for example, from a blue-green alga to a non-photosynthetic bacterium. The phenomenon of HGT is not only of an interest to the science of molecular genetics and biology, but also to the biosafety issue of genetic engineering. The novel protocol reported here for the first time teaches how to measure HGT from a genetically engineered (GE) blue-green alga (gene donor) to wild-type •According to this novel protocol, the HGT frequency may be calculated from the number of HGT recipient colonies observed, the number of recipient cells plated, and the donor-recipient co-incubation time.•This approach can also help test the possible HGT routes to assess whether a HGT is through a direct cell-to-cell interaction or by an indirect cell-to-liquid environment-to-cell process.•The protocol may be applied in full and/or in part with adjustments to measure HGT for a wide range of donor and recipient organisms of interest. Specifications Table•Thermosynechococcus elongatus BP1 or other blue-green algae (cyanobacteria) such as Synechocystis sp. strain PCC 6803 as a transgene(s) donor for horizontal gene transfer (HGT) assay.Genetically engineered (GE) •Escherichia coli strain such as E. coli DH5α or the other non-photosynthetic bacteria to serve as a transgene(s) recipient for horizontal gene transfer (HGT) assay.Wild-type •0SA cyanobacteria culture media.BG-11 and BG-11•Antibiotics .•High-grade sterile LB agar plates with antibiotic and without antibiotic (control).•Photosynthetic actinic light sources such as daylight fluorescent lamps.•LI-COR LI-250A light meter.•Properly autoclaved culture plates/tubes/flasks for cell growth.•Temperature-controlled shaking incubators (IKA KS 4000 i control) for culture growth.•Innova 2300 platform shaker.•Olympus DP672 Microscope.•Benchtop centrifuge (Eppendorf Centrifuge - 5418 R) for DNA preparation.•Beckman Coulter Avanti J-26 Xp cooling centrifuge for cell harvesting and DNA preparation.•Spectrophotometer for monitoring cell growth.•Erlenmeyer flask of different size depending on the culture volume.•Plasmid vectors such as pUC57-based pKB plasmid containing designer DNA constructs (transgenes) of interest including antibiotic selectable marker.•BioRad C1000 thermal cycler for PCR.•Thermoscientific nanodrop 2000 spectrophotometer for DNA quantification.•UVP Benchtop UV transilluminator/PhotoDoc-it Imaging System.•Gene pulser Xcell Electroporation BioRad .•Thermoscientific Heratherm incubator IGS 100.•Percival Environmental Control Growth Chamber/Incubator.•Yamato Autoclave/ Sterilizer SM510.•Class II biosafety cabinet .•Note: This list does not include any small generic laboratory equipment that are assumed to be available. Chemicals and other components can be used from any reliable company.•E. coli and blue-green algae such as Thermosynechococcus elongatus BP1 hosts.Choice of appropriate antibiotics depends on the vector construct, bacteria such as •E. coli and Thermosynechococcus elongatus BP1 strains are commercially available in the market; they can also be used for molecular cloning and horizontal gene transfer (HGT) assay as per the recommended protocol with appropriate selective antibiotics.Note: Several other vectors, 1Prepare special plasmid containing designer transgene(s) of interest including an antibiotic selectable marker to create genetically engineered (GE) cyanobacteria to use as a transgene(s) donor for the horizontal gene transfer assay .2T. elongatus BP1) through genetic transformation using electroporation followed by transformant colony selection and verification with PCR.Create special plasmid-based and/or genomic-based GE cyanobacteria  by centrifugation using Beckman Coulter Avanti J-26 Xp centrifuge.Harvest photoautotrophically grown GE 6E. coli cells from actively growing liquid culture (at mid logarithm-growth phase) by centrifugation using Beckman Coulter Avanti J-26 Xp centrifuge.Harvest wild-type 7T. elongatus BP1 in BG-110SA liquid medium at the cell population density of about 107 cells/mL.Make cell suspension of GE 8E. coli DH5α in LB liquid medium at the cell population density of about 107 cells/mL.Make cell resuspension of wild-type 9T. elongatus BP1 (donor) cells resuspension (in BG-110SA) with 15 mL of wild-type E. coli DH5α (recipient) cells resuspension (in LB).Mix 15 mL of GE 10Incubate the two (donor and recipient) organisms at 1:1 population ratio (the “15 mL + 15 mL” mixture) on a shaker at 37 °C under continuous photosynthetic light intensity provided by daylight fluorescent lamps for a designated period of time (11Take samples from liquid incubation co-culture and plate the liquid co-culture samples on LB agar medium in the presence and absence (control) of antibiotic; and spread (plate) a designated volume  colonies (Count the HGT events by counting the number of 14E. coli colonies (cells) by observation using Olympus DP672 Microscope in combination with control experiments.Verify the HGT 15E. coli colonies by colony PCR in combination with control experiments.Verify the HGT 16E. coli) colonies observed per antibiotic LB agar selective plate, the volume (E. coli) cells in the co-incubation liquid, and the donor-recipient co-incubation time  frequency from the number  for creation of GE cyanobacteria including GE T. elongatus BP1 (pKB plasmid transformant) has been experimentally demonstrated in the Lee Laboratory at Old Dominion University and recently reported in Ref. [To measure HGT in accordance of this protocol, it is a preferred practice to use genetically engineered cyanobacteria  with a selectable marker such as an antibiotic-resistant transgene as a transgene donor. Currently, genetically engineered (GE) cyanobacteria can be created through two types of genetic transformation: 1) plasmid-based genetic transformation and 2) integrative genetic transformation through homologous recombination into the host genomic DNA. In a plasmid transformant, the transgenes such as an antibiotic-resistant transgene are in a self-replicable plasmid like pUC57 that can somehow replicate also in certain host cyanobacterial cell. The copy number of the plasmid per cell may vary depending on how well the plasmid could replicate in the cyanobacterial cell. During cyanobacterial cell division, the distribution of plasmid to the daughter cells may be random in nature. Consequently, plasmid transformants tend to be less stable than the genomic transformants whose transgenes are integrated into their genomic DNA. It is likely that the transgenes carried by a plasmid may be more mobile for HTG than those integrated into the cyanobacterial genomic DNA. The protocol may be used to test this feature by measuring HGT frequency for both the plasmid transgenes and/or genomic transgenes from GE cyanobacteria to T. elongatus BP1 (pKB plasmid transformant) cells were photoautotrophically grown at 45 °C in a Percival growth chamber in BG-110SA medium containing kanamycin (40 μg/ml), and wild-type E. coli DH5α cells were grown in LB broth at 37 °C in a shaking incubator. The photoautotrophically grown cells of GE T. elongatus BP1 were collected by centrifugation. The supernatant was discarded and the cells were resuspended in fresh BG-110SA medium without antibiotic. Both GE T. elongatus BP1 cells and wild-type E. coli cells were resuspended to population density of about 107 cells/mL  and 15 mL of LB broth without wild-type E. coli DH5α cells. Control 2 consisted of 15 mL of wild-type E. coli DH5α (107 cells/mL) and 15 mL of BG-110SA medium without T. elongatus BP1 transformant. These controls were also incubated in a shaking incubator at 37 °C under the same lighting conditions. Samples from each liquid co-culture and control were collected after 1, 2, and 3 days and were then sampled and spread on control LB agar plates and selection LB plates containing kanamycin. Typically, about 100 μl of liquid incubation co-culture sample was used to spread onto the surface of a 45 mL antibiotic-containing LB agar medium per petri dish  plate (–2 s–1. PCR was used to verify the presence of the transgene cassette DNA within these colonies [Using the cells resuspension at a concentration of 10p) plate . The LB T. elongatus BP1 (pKB plasmid transformant) has ability to transfer its pKB plasmid into wild-type E. coli DH5α, the two (donor and recipient) organisms were co-incubated at 1:1 population ratio for a designated period of time [T. elongatus BP1 transformant, showed no colony growth on LB plates with and without kanamycin after 48 h of plate incubation at 37 °C under lighting. This indicated that T. elongatus BP1 could not grow on LB plates. Therefore, any colony formed on LB agar plates from this experiment would be E. coli. Bacterial growth was seen on LB plates without antibiotic for control 2 which contained wild-type E. coli only. However, control 2 did not have any colony growth on LB plates with kanamycin (T. elongatus and wild-type E. coli liquid incubation co-cultures). Typically, the observed number of HGT E. coli colonies ranged from 20 to 75 per plate [T. elongatus BP1 growing on LB agar plates, the colonies on LB plates with kanamycin were indeed E. coli  and recipient (E. coli) cells in the co-incubation liquid and to see the cells from selected HGT E. coli colonies. T. elongatus BP1 transformants) and recipient (E. coli DH5α) cells in the co-incubation liquid that was examined under a light microscope (Olympus DP672 Microscope) after 2 days of co-incubation. E. coli DH5α cells from a colony obtained from LB kanamycin plate of co-culture sample containing T. elongatus BP1 transformants and E. coli DH5α. These microscopic examination results indicate that the protocol is working since the cells from the selected HGT recipient colonies were indeed the HGT E. coli DH5α cells as expected [The techniques of microscopic imaging can certainly be employed to visualize the donor (E. coli) colonies observed per antibiotic LB agar selective plate, the volume (E. coli) cells in the co-incubation liquid, and the donor-recipient co-incubation time  colonies per antibiotic LB agar plate under the given experimental conditions including the plated co-incubation liquid cell suspension volume of 100 μl, recipient cell concentration of 1/2 × 107E. coli cells /1000 μl in the co-incubation liquid, and the donor-recipient co-incubation time of 2 days. The HGT frequency (probability) from the plasmid transformants of cyanobacterium Thermosynechococcus elongatus BP1 (donor) to wild-type E. coli (recipient) was calculated to be in a range from 2.0 × 10−5 to 7.5 10−5 per cell day. That is, there could be about 20 to 75 HGT events per million recipient (E. coli) cells daily from the plasmid-based GE blue-green alga Thermosynechococcus under the given experimental conditions.As a conclusion, the HGT frequency can be calculated from the number of HGT recipient colonies observed, the number of recipient cells plated, and the donor-recipient co-incubation time. This approach can also help test for the possible HGT routes to assess whether a HGT is through a direct cell-to-cell interaction or by an indirect cell-to-liquid environment-to-cell process. The protocol may be applied in full and/or in part with adjustments to measure HGT for a wide range of donor and recipient organisms of interest, for example, from algae to non-photosynthetic organisms.1Thermosynechococcus elongatus BP1 to grow on LB agar plate makes the counting of HGT E coli colonies quite convenient. However, the protocol may be used for all cyanobacteria including those that can grow on LB agar plate as well, since cyanobacterial colonies typically have a blue-green color readily distinguishable from E coli colonies.The inability for certain cyanobacteria such as 2E. coli) cells. Sometimes, it may be challenging to find a liquid culture medium that could accommodate both the donor (cyanobacteria) and recipient (E. coli) cells. A logical solution may be a 50–50 mixture of the donor liquid culture medium (such as BG-110SA) and the recipient liquid culture medium (such as LB) as used in the present experiment.It is important to properly select the HGT co-incubation liquid medium, which should be able to accommodate both the donor (cyanobacteria) and recipient (3E. coli) cells should be performed under proper experimental conditions including not only the temperature but also the lighting conditions to accommodate both the donor and recipient cells in consideration of a natural environment such as in case of GE cyanobacteria accidently released into a natural pond environment, where they may meet with other bacteria like E. coli. The light intensity typically needs to be at least about 5 μE m–2 s–1 to ensure the blue-green algae being able to function properly with photosynthetic energy support.The liquid co-incubation of the donor (cyanobacteria) and recipient  for HGT 5E. coli) in the co-incubation liquid, but not that of the donor (GE cyanobacteria). Therefore, in a “15 mL + 15 mL” GE T. elongatus and wild-type E. coli co-incubation liquid, the value for E. coli cell stock concentration (107E. coli cells/1000 μl) used in making the 50-50 co-culture of GE T. elongatus and wild-type E. coli.Note, the 6Based on the protocol, the donor of genetic material for HGT assays does not have to be GE cyanobacteria. It can be a plasmid material such as the pKB plasmid tested in the experiment . It can 7The protocol may be used to also test for certain possible routes of HGT such as to answer the questions of: 1) Does a HGT require a direct cell-to-cell interaction? or 2) Could a recipient cell pick up a piece of DNA in a liquid environment such as a plasmid that may be released from a donor cell (an indirect cell-to-liquid environment-to-cell process for HGT)? This type of questions can be answered by comparative HGT assays in presence and absence of GE cells and/or free plasmid DNA in incubation liquid as demonstrated in the experiments of 8T. elongatus BP1 to wild-type E. coli was estimated here to be in a range from 2.0 × 10−5 to 7.5 10−5 per cell day. The HGT probability from the genomic transformants of cyanobacteria to wild-type E. coli is likely to be rather low, which is yet to be measured. Special efforts such as increasing the incubation time and the donor and recipient cell population density for HGT liquid co-incubation with multiple replications may be needed to measure for such a potentially low probability genomic HGT event. This type of potentially challenging experiments with multiple replications may take months and even years of efforts to accomplish. It is a preferred practice to run a positive control experiment measuring HGT from a well-characterized plasmid transformant such as the pKB plasmid T. elongatus BP1 transformant to E. coli as a control in parallel with the long-duration main assay experiments to measure the genomic HGT event. This may help to ensure the long-duration experimental setups and conditions are all properly working so that the ultimate assay result whether positive or negative will represent the true outcome from the genomic HGT process of interest.Based on the present experimental result , the HGT9E. coli. Other recipient organisms of interest such as yeast and human health-related bacteria including (but not limited to) Shigella, Campylobacter, and Salmonella may also be used for HGT assays in accordance of the protocol. Therefore, both the co-incubation liquid media and selective agar media plates may be adjusted in accordance of the protocol for the HGT assays based on the specific donor and recipient organisms of interest.This protocol may be adjusted and applied in full and/or in part in combination with adjustments to measure HGT for a wide range of donor and recipient organisms from algae to non-photosynthetic organisms. For example, the recipient cell does not necessarily have to be E. coli.In contrast to the "vertical" transmission of DNA from the parent to its offspring of the same species, horizontal gene transfer (HGT) is a natural process for an organism to transfer genetic material to another organism that is not of the same species, for example, from a blue-green alga to a totally distinct non-photosynthetic bacterium. It is generally believed that HGT is used as a means of evolution . The phe2) in the Earth’s atmosphere and their inevitable depletion as the world’s major fuel source [2), decrease in competition for land and crops, and potential in alleviating the increase of CO2 in the atmosphere by being net carbon neutral [The phenomenon of HGT is not only of an interest to the modern science of molecular genetics and biology, but also to the biosafety issue of genetic engineering in relation to biofuel energy and environmental sustainability as well as public health and wellbeing. Synthetic biology using genetically engineered (GE) cyanobacteria has the potential to produce valuable products such as biofuels. In recent years, GE cyanobacteria have become a promising new agriculture avenue for photosynthetic renewable energy. This approach addresses concerns of fossil fuels including their contribution to the increase in greenhouse gases  will advance basic science knowledge in synthetic biology with microorganisms and could assist government agencies in the development of important policies and regulations for genetic engineering and use of GE cyanobacteria for biofuel production as well as other areas.Thermosynechococcus elongatus BP1 has the potential to transfer its transgenes to wild-type E. coli and assess their possible HGT frequency. Earlier studies of HGT showed that E. coli can transfer genes to cyanobacteria [E. coli was experimentally demonstrated through the use of GE T. elongatus BP1 carrying a designer DNA construct with a plasmid to produce alcohol that was co-incubated with wild-type E. coli DH5α in a 1:1 ratio. HGT was then monitored on solid Luria Broth (LB) media with selective antibiotic for E. coli growth. The experimental results indicate that plasmids can be transferred from GE T. elongatus BP1 (transgene donor) to wild-type E.coli (recipient) after two days of liquid co-incubation [Therefore, this protocol is developed to determine whether a blue-green alga such as GE bacteria . In the Escherichia coli or other bacteria containing recombinant DNA transgenes and cyanobacteria (HGT in the opposite direction). Another possibility is to use an E. coli strain carrying a plasmid with a selectable marker different from that present in the cyanobacterial donor in this experimental scheme, then to subsequently screen for both selectable markers following the co-incubation step. Therefore, this novel protocol may be adjusted and applied in full and/or in part in combination with adjustments to measure HGT for a wide range of donor and recipient organisms from algae to non-photosynthetic organisms, which could be highly useful for a number of applications in various fields from the science of molecular genetics and biology to the biosafety of genetic engineering.In summary, this protocol could be extremely useful for testing of HGT between diverse microorganisms employing recombinant transgene DNA constructs. It utilizes the intrinsic differences in appearance and metabolism, with selectable antibiotic markers, to specifically screen for the transfer of gene(s) between transgenic blue-green algae and non-photosynthetic bacteria. Presence of the transgene(s) in the putative HGT recipients is then confirmed by PCR in combination of colony formation and re-streaking on culture plates with antibiotics. This type of assay may provide quantifiable information on possible unintentional genetic transfer from recombinant microalgae developed for commercial production to other microorganisms present in the environment. This approach could also be used to estimate horizontal gene transfer between Gene, 704:49–58 [The author JWL conceived the original idea of measuring horizontal gene transfer from algae to non-photosynthetic organisms and wrote this article for the novel protocol here with his original equation on HGT Frequency calculation. JWL first proposed the idea and wrote the method for measuring horizontal gene transfer in a research grant application in 2015. The proposed project was subsequently funded by the Biotechnology Risk Assessment Grant Program competitive grant award no. 2016-33522-25624 from the U.S. Department of Agriculture. This method has now been successfully practiced experimentally through the funded project work activity in measuring horizontal gene transfer. The graphic abstract was adapted and modified from Nguyen et al. 2019


The aim of this study was to analyze the current status and problems of hearing screening tests for newborns in low-income families in the southeastern Korea.This study analyzed data from the Ministry of Health and Welfare’s project on the early detection of hearing loss in newborns in low-income families in the southeastern Korea (2011-2015).The referral rate was 1.33, 1.69, and 1.27% in Daegu, Gyeongbuk, and Ulsan, respectively. The confirmatory test rate was 36.09, 23.38, and 52.94% in Daegu, Gyeongbuk, and Ulsan, respectively. The incidence of hearing loss (adjusted) was 0.41, 0.62, and 0.41% in Daegu, Gyeongbuk, and Ulsan, respectively. After confirming hearing loss, newborns with hearing handicaps were mostly lost to follow-up, and rehabilitation methods, such as hearing aids or cochlear implants, were not used. The screening tests were performed within 1 month of birth, and the confirmatory tests were generally performed within 3 months of birth. However, more than 3 months passed before the confirmatory tests were performed in infants with risk factors for hearing loss in Gyeongbuk and Ulsan.Hearing screening tests were conducted in newborns from low-income families in southeastern Korea who received a coupon for free testing, but the newborns that were referred after the screening tests were not promptly linked to the hospitals where confirmatory tests were performed. Furthermore, hearing rehabilitation was not consistently performed after hearing loss was confirmed. To successful early hearing loss detection and intervention, a systematic tracking system of hearing loss children is needed. Hearing loss is one of the most common diseases in newborns. Three to five out of every 1,000 newborns suffer from some form of congenital bilateral hearing loss above 40 decibels (dB), and 1-2 out of every 1,000 newborn babies have severe hearing loss . HearingA prospective study was performed on 150 hearing-impaired newborns who began hearing rehabilitation within 2 months after the detection of hearing loss. The children were divided into 2 groups: (A) newborns with hearing loss detected before 6 months of life; and (B) newborns with hearing loss detected after 6 months. The children in group A had more advanced language development than those in group B. Therefore, for effective hearing development and rehabilitation, hearing loss should be diagnosed by at most 6 months after birth, and treatment for hearing impairment should be initiated as soon as possible .Newborn hearing screening tests have been regularly conducted in developed countries since 1990 and were introduced in Korea in the 2000s. The type of hearing loss targeted by these tests is permanent unilateral or bilateral conductive or sensoryneural hearing loss, including auditory neuropathy, averaging 30-40 dB or more  . All newApproximately 50% of newborns and infants with hearing loss have at least 1 risk factor for hearing loss, with the remaining 50% having no risk factors. Currently, most developed countries conduct hearing screening tests on all newborns . StartinBefore the implementation of health insurance coverage for hearing screening tests on all newborns in Korea, the current status of designated hospitals and each region regarding hearing screening tests should be examined and analyzed, and any problems detected should be corrected. Thus, an appropriate level of testing should be carried out irrespective of the region.This study analyzed hearing screening data from newborns in low-income families in the southeastern region that received support from the Korean Ministry of Health and Welfare. The current status and problems of newborn hearing screening in these infants were assessed to obtain information relevant for the quality control of newborn hearing screening tests when universal hearing screening of all newborns is performed at the national level.This study analyzed the southeastern region of Korea  from 2011 to 2015, using the data set of 2the Ministry of Health and Welfare’s project on the early detection of hearing loss in newborns in low-income families in 17 cities and provinces. The distribution and status of the designated screening hospitals were analyzed from 2013 to 2015.The project was carried out as follows . The carThe following evaluation indices were examined. The screening rate was the percentage of newborn babies screened in a given region. The referral rate was the percentage of newborns who did not pass the hearing screen on either 1or both ears and were referred for a hearing confirmatory test among the newborns who were screened. This was preferably less than 4% of healthy newborns . The typThe study protocol was approved by the institutional review board (IRB) of Dongguk University Gyeongju Hospital (IRB no. 110757-201805-HR-07-02).In 2015, 41 OBGY clinics, 10 ENT clinics of general hospitals, and 2 private clinics participated in this project in the southeastern region . In the southeastern region from 2011 to 2015, 274,222 newborns were involved, 35,100 coupons were issued, and 33,108 newborns were screened. The screening rate was 12.07%, the referral rate was 1.50%, the confirmation rate was 31.85%, and the prevalence of hearing loss (after correction) was 0.48%. The average time from birth to screening was 4.65 days and the time from birth to confirmation was 72.21 days. Of the screening tests, 84.51% were performed by AABR and the expedition fertility rate was 13.56%. The screening rate was 9.96, 14.22, and 11.45% and the referral rate was 1.33, 1.69, and 1.27% in Daegu, Gyeongbuk, and Ulsan, respectively (p<0.05). The confirmatory test rate was 36.09, 23.38, and 52.94% in Daegu, Gyeongbuk, and Ulsan, respectively. Gyeongbuk showed a significantly lower confirmatory test rate than the other regions (p<0.05). The prevalence of hearing loss after correction was 0.41, 0.62, and 0.41% in Daegu, Gyeongbuk, and Ulsan, respectively (p>0.05). The severity of hearing loss was different in each region (p<0.05). A difference in to the types of early intervention in each region was observed. Loss to follow-up was the most common outcome in all 3 regions. Follow-up was more common in Gyeongbuk than in the other regions, but this tendency was not significant. The time from birth to screening was 3.27-6.11days and the time from birth to confirmation was 68.95-75.78 days in these areas. AABR was performed more than AOAE in all 3 regions. AABR was used significantly more in Daegu than in the other regions (p<0.05). The expedition fertility rate of Gyeongbuk was significantly higher than that of the other regions (p<0.05). Of the mothers who resided in Gyeongbuk in 2015 but delivered in another region, 81.46% delivered in Daegu .In 2015, 15 OBGY clinics, 5 ENT clinics at general hospitals, and 1 private clinic participated in the hearing screening project in Daegu; 19 OBGY clinics, 4 ENT clinics at general hospitals, and 1 private clinic in Gyeongbuk did so; and 7 OBGY clinics, 1 ENT clinic at a general hospital, and no private clinics did so in Ulsan.The data were reviewed according to the type of institution designated for screening from 2013 to 2015 . In eachIn the southeastern region from 2011 to 2015, 3.53% of applicants were high-risk newborns, as were 9.80% of newborns with hearing loss . FurtherAlthough some cities in Gyeongbuk had multiple screening hospitals, there were many cities and counties (guns) with 1  or no screening hospitals . In GyeoInitially, this study comprehensively reviewed the status and problems of hearing screening tests of newborns in low-income families in the southeastern region of Korea.More than 95% of all newborn babies should be screened in the first month of life . BecauseMore than 90% of the newborns referred by screening should receive a confirmatory test within 3 months of birth . The conAural rehabilitation, such as hearing aids, after confirmed hearing loss should be started within 6 months of birth . None ofIn this study, 4-6 of every 1,000 newborns from low-income families had hearing loss, which was similar to the rates reported in other studies . In thisScreening by ABR was significantly more common than screening by AOAE. AABR was used much more often in Daegu than in the other regions . AABR apThe problems regarding newborn hearing screening in each region were also reviewed. Considering the current situation in Daegu, the referral rate of OBGY clinics, which accounted for the largest number of primary screening institutions, was very low (0.72%). The number of tests performed on each ear at a time should be checked, especially at OBGY clinics in Daegu. Gyeongbuk had a high expedition fertility rate and did not have a constant distribution of screening institutions; there were no screening hospitals in 2 cities (Mungyeong and Yeongcheon) and 12 counties . No hospitals offering confirmatory tests were in the vicinity of Uljin, Yeongyang, and Yeongdeok-gun in the north-eastern part of Gyeongbuk . GyeongbIn Ulsan, 6.69% of the newborns who were screened were at high risk, which was significantly higher than the corresponding rate in Daegu or Gyeongbuk. Fortunately, the confirmatory test rate was 52.94%, which showed that the linkage of newborns requiring a referral to the confirmatory test was better in Ulsan than in the other 2 regions, and the probability of missing a hearing-impaired high-risk newborn was somewhat lower. Efforts should be made to determine why there were more newborns with high-risk factors in Ulsan.This study had some limitations. The results of this analysis alone may not be consistent with the results of hearing screening tests of all newborns in those areas in the future because this study analyzed data from the Health and Welfare Ministry’s project that provided support for the early diagnosis of newborn hearing loss in low-income families (2011-2015). For example, in low-income families, it is more likely that the mother and/or newborn will have poor health or nutritional status or be exposed to an environment harmful for hearing or to sources of infection. Additionally, purchasing hearing aids imposes an economic burden on low-income families. Therefore, the prevalence of hearing loss and the percentage of newborns at a high risk of hearing loss might be higher in the low-income families included in this study than in the overall population, and the early intervention rate after confirming hearing loss might be lower.In conclusion, newborn hearing screening in low-income families in Daegu, Gyeongbuk, and Ulsan was implemented to some extent. However, most newborns who received a referral did not undergo a hearing confirmatory test. In addition, even if hearing loss was diagnosed, early interventions such as hearing aids or cochlear implants were not provided, meaning that newborns with hearing loss did not receive the appropriate help for hearing development. In particular, in newborns at high risk for hearing loss, a considerable time passed from birth to the confirmatory test. Therefore, quality control of testing in high-risk newborns is necessary.


Worldwide approximately 360 million people suffer from hearing impairment, 328 million of whom are adults. Up to now there has been no systematic evaluation of any representative epidemiological data on the prevalence of hearing loss among adults in Germany. The present paper is intended to investigate this within the framework of a systematic review.A systematic literature search was carried out in electronic databases as well as by means of hand-searching. Studies published after 1975 and indicating the prevalence or incidence of hearing impairment among German adults were included. Study selection, data extraction and additional quality assessments were made by two independent reviewers.By means of a systematic literature search it was possible to identify 6 sources, which provided solely cross-sectional data, whereby the reported data are based on a study population of between some hundred and 10 million people living in Germany. The prevalences ascertained showed a broad range of between 16% and 25% and varied according to age, study setting, definition of hearing loss and method of data capture. At present there are no utilizable data on the extent of the use of hearing aids.The present review demonstrates clearly that evidence-based information relating to Germany can only be made on the basis of a clear definition of hearing loss within the framework of an up-to-date and representative epidemiological study carried out with appropriate methodology. In view of the high prevalence of illnesses causing hearing impairment and of the risks to health associated with untreated hearing impairment as well as of socio-economic costs, such an epidemiological study is of great social significance.The online version of this article (10.1007/s00405-019-05312-z) contains supplementary material, which is available to authorized users. According to the Global Burden Disease Study of the World Health Organization (WHO), hearing impairments are one of the most common disorders in patients reducing the quality of life in most industrialized countries . WorldwiBesides vascular disorders, chronic inflammation, noise exposure, genetic susceptibility, and tumors (such as vestibular schwannoma), the physiological aging of the inner ear is often the cause of hearing impairments at ages over the fifth or sixth decade of life. This aging process is referred to as presbycusis . It devehttp://www.who.int/news-room/fact-sheets/detail/deafness-and-hearing-loss). Although disorders associated with hearing losses are regarded as a significant medical problem, undertreatment is common, especially in the case of presbycusis due to its slow progression. For example, it was reported that only 16% of those requiring treatment are receiving an adequate intervention in form of hearing aids . We also searched the bibliographies of relevant studies to identify further citations manually. A search was also made for ongoing or completed but not yet published studies in the registers for clinical trials  and the German study register  is presented in the Online Appendix. Search strategies for other databases were modified to meet the requirements of each database.Two reviewers  screened the titles and abstracts of all reports identified by electronic searches and hand searching. Afterward, we obtained full-text copies of all potentially relevant articles and again, two reviewers  assessed them for inclusion.outcome data, i.e., details of prevalence and / or incidence of the measured hearing impairment as well as information on the proportion of people with hearing impairments treated with hearing aids.The above-mentioned authors also independently carried out data extraction and performed the methodological assessment of the included studies. Any discrepancies in the data extraction were resolved by discussion. The following data were extracted from the studies: (i) details of publication characteristics, i.e., bibliographical data , year of publication), information on the design of the study, exact geographical area of data collection, period of data collection, age and number of adults included, and the definition of the measured hearing impairment (including the assessment method); (ii) The risk of bias  and generalizability  was assessed according to pre-defined criteria which were developed by our group considering published epidemiological literature  and inteFor data extraction and the assessment of the risk of bias and generalizability of results, we relied on information provided in the individual studies.n = 16) and the DRKS register (n = 18)]. After deducting the duplicates, there remained 2111 references. These references were assessed on the basis of their title and abstract. In total, 1948 references were excluded because they did not address our research question. Finally, 163 potentially relevant references were included for full-text screening. From these, six studies (10 publications) provided data on the prevalence of hearing disorders. The study flow is presented in Fig. The systematic literature search identified 2478 references. Additionally, we identified 53 references by hand searching, including 36 registry entries referring to ongoing or completed and not yet published studies [clinicalstrials.gov n = 6 and theTable The study identified in the DRKS register (HörMAT) limited the age of the included population and included adults between 50 and 75 years of age . The remThree studies gathered their data throughout Germany , 25, 26,The RKI reported prevalence data based on a telephone survey  . The remData on the prevalence of hearing loss are shown in Table Only two studies provided data on the prevalence of hearing aids in Germany , 20. HÖRThe methodological assessment is shown in Fig. The nationwide health survey carried out by the German RKI is susceptible to a high risk of bias as no objectively measurable threshold values can be taken into account with this subjective self-assessment by telephone . The remData for the entire population of Germany were provided by three studies , 25, 26.Only a limited number of studies are available providing prevalence estimates of hearing impairment for the general population in Germany. Moreover, the current epidemiological data are either associated with risk of bias or lacking generalizability. Overall, prevalence data on hearing impairment for the German population showed a wide variety ranging between 16 and 25% depending on the age, the study setting, the definition of hearing impairment and the method how hearing impairment was measured. For example, the reported hearing impairment was either based on (i) frequency-specific measurements by PTAs , (ii) onFive primary studies claim that their data are representative for the entire population living in Germany , 25, 26,An estimate of the magnitude of these unrecorded cases may be provided by studies comparing prevalence data obtained by subjective self-assessments and making use of PTAs , 20, 25.We identified only two studies reporting on treatment coverage. However, none of these studies allowed us to judge the proportion of patients not wearing hearing aids although it would be indicated , 20. TheIn the ongoing German National Cohort (GNC) men and women aged between 20 and 69 are clinically examined in 18 study centers throughout Germany . In totaOverall, an epidemiological systematic review provides a useful approach to capture prevalence data on a certain clinical condition. However, some challenges in relation to the methodological assessment of the primary studies with respect to the risk of bias and the generalizability of the results exist. For example, there are no well-established tools to estimate the methodological quality of such studies. Therefore, based on published epidemiological studies we developed criteria to assess both risks of bias and generalizability for the included studies which may be of high value in future epidemiological research . FurtherThis work has implications for researchers and those who use epidemiological data to help inform clinical and policy decisions. Overall, we revealed that (i) prevalence data on hearing loss are scarce and show methodological flaws. (ii) Prevalence data were broadly scattered reflecting different definitions of hearing loss, different methods of data capture, different not comparable age-groups and different settings; therefore, a comparison between studies was challenging. Further studies should be done by on the basis of a clear definition of hearing loss. This definition ought to be worked out in a first step. (iii) Moreover, available data are either not representative for the general in Germany living adult population  or show methodological flaws limiting their validity. (iv) The rate of patients with hearing loss wearing hearing aids is very low Fig. . AlthougBelow is the link to the electronic supplementary material.Supplementary material 1 (DOCX 22 KB)


Context: Limonoids possess broad range of biological activities, including antitumour, antimicrobial and antioxidant activities, etc. Eudesmin (EDN) is a type of limonoid which also possesses various activities. However, there is no report on the antitumour lung cancer (LC) activities of this compound.Objective: The present study investigates the antitumour effects of EDN and its potential molecular mechanisms.Materials and methods: The in vitro antitumour effects of EDN on LC A549 cells were evaluated by using MTT assay. The in vivo antitumour effects were investigated on a xenograft athymic nude mouse model. The mice were administered orally with EDN  once daily for 28 days. Effects of EDN on apoptosis-related or signalling proteins  were assayed by western blot analysis.Results: EDN showed significant inhibitory effects on the growth of LC A549 cells in vitro with the half maximal inhibitory concentration (IC50) of 18.3 μM. By treating with EDN , expression of caspase-3, caspase-9, Bax, P53 and phosphorylated JNK in A549 cells were significantly upregulated, whereas expression of Bcl-2 and Akt phosphorylation were significantly down-regulated. Interestingly, EDN-induced apoptosis could be attenuated by JNK inhibitor. In addition, in vivo experiments also indicated EDN  had significant antitumour effects (p < 0.01) on nude mice.:ConclusionsOverall, the results indicated that EDN possesses significant antitumour effects on LC and the possible mechanism might be related to induction of mitochondria-mediated apoptosis. Worldwide, lung cancer (LC) is one of the most commonly occurring cancers in humans, there are more than one million deaths every year  was obtained from Chengdu Herbperity Co., Ltd . 1--3,5-diphenyl-formazan (MTT) was purchased from Sigma-Aldrich . The primary monoclonal antibodies of P53, Bcl-2, Bax, caspase-9, caspase-3, Akt, JNK and horseradish peroxidase-conjugated secondary antibodies were obtained from Beyotime Biotechnology . RPMI1640 culture medium and foetal bovine serum (FBS) were purchased from American Hyclone Company . All other chemicals and reagents used in this study were of analytical grade.2/95% air.Human lung carcinoma A549 cells were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . They were maintained in RPMI-1640 culture medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37 °C in 5% CO4 cells/mL and incubated overnight. Then, the cells were incubated with different concentrations of PSDP  for 24 h. MTT  was added to each well and cultured for 4 h. After MTT solution was removed, DMSO (150 μL) was added and oscillated for 15 min. The absorbance was measured at 570 nm using a microplate reader . The inhibition of cell proliferation was calculated by the following formula:A0 is the absorbance of the control group and At is the absorbance of EDN treated group.The effect of EDN on A549 cell proliferation was assayed by using MTT assay according to a previous study  guidelines and the experiments were approved by the Animal Ethics Committee of Linyi People's Hospital .Male athymic nude mice (nu/nu) aging 4–6 weeks (18 ± 1 g) were purchased from SLAC Laboratory Animal Co., Ltd. . All mice were housed in a temperature of 21 ± 2 °C, relative humidity (65 ± 10%) and a 12 h light/dark cycle environment, and fed with food and water 6/0.2 mL) into the dorsal flank of the mouse : the control group (distilled water), different EDN groups . The mice were then administered orally once daily for 28 days, and tumour volumes were measured by digital calliper during this period. Tumour volume was calculated by a standard formula: (width2×length)/2. After 24 h of the last administration, the mice were sacrificed, and the tumours weights and body weights were measured.Subcutaneous tumour model was established in athymic nude mice by inoculation of A549 cells . Differences of groups were considered statistically significant when p < 0.05.All data were expressed as means ± standard deviation (SD). Statistical analysis was performed by Student’s in vitro antitumour activity of EDN was assayed by MTT in a concentration range from 2.5 to 80 μM. As a result, EDN showed significant inhibitory effect on the growth of A549 cells with the increase of concentration. The half maximal inhibitory concentration (IC50) of EDN on A549 cell growth was 18.3 μM. Thus, the concentrations of 10, 20 and 40 μM were selected for further experiments.As shown in The effects of EDN on the expression of P53, Bcl-2, Bax, caspase-9, caspase-8 and caspase-3 proteins in A549 cells are shown in To investigate whether the pro-apoptosis effects of EDN through the cell JNK/Akt signal pathway, the expression of Akt and JNK proteins in A549 cells was evaluated. As shown in To further determine whether regulation of JNK was required in EDN-induced apoptosis, the A549 cells were treated with 40 μM EDN in the presence or absence of the JNK siRNA. The results are shown in p < 0.01) at the doses of 10, 20 and 40 mg/mL compared with the mice in control group. However, EDN showed no significant effect on the body weight of mice (p > 0.05) after oral administration for 28 days compared with the control mice, indicating that EDN had no obvious toxicity.The effect of EDN on tumour weight and the body weight in A549 cells transplanted mice is shown in in vivo, and also confirmed that the EDN-induced apoptosis is associated with the mitochondria-mediated apoptosis pathway.The effects of EDN on the expression of P53, Bcl-2, Bax, caspase-9, caspase-3 and JNK in tumour tissues are shown in 6–C3 carbons are bound by the C8 central carbon. They are widely distributed in more than 70 families of vascular plants and have been isolated from different parts of plants, such as roots, stems, rhizomes, leaves, seeds and fruits, etc. (Jiang et al. in vitro and in vivo, demonstrating that it had the potential to develop into antitumour drugs for LC in the future.Lung cancer is an intractable disease and still remains a leading cause of cancer death in the world. However, current antitumour agents often have serious side effects (Park et al. Apoptosis, known as programed cell death, has been recognized as an ideal method of cancer therapy, and is controlled by genes and a series of enzymes (Tai et al. It is reported that the MAPK pathway plays important roles in cell proliferation, apoptosis and differentiation, and can be activated by several stimuli (Gao et al. in vitro and in vivo. The mechanisms of the antitumour effects might be closely related to the induced apoptosis via mitochondria-mediated pathway in the tumour cells. EDN has potentials to be developed into antitumour drugs for the treatment of LC.In conclusion, the present study proved that EDN possessed marked antitumour effects on LC A549 cells both


Objectives: This study aims to understand the disability status of the elderly residents of a County  and explore its influencing factors. Methods: Respondents consisted of 2300 elderly people aged 60 and above from three townships in the county we studied. The Activities of Daily Living (ADL) Scale was used to assess the disability of the elderly sample. Chi-square test was applied to compare the disability rate among the elderly with different demographic characteristics. The graph showed the disability rates of ADL, six items of Physical Activities of Daily Living (PADL) and eight items of Instrumental Activities of Daily Living (IADL) at different ages. Binary logistic regression was used to analyze the influencing factors of disability rate among the elderly. Results: The disability rates of ADL, PADL, and IADL in the elderly were 43.4%, 11.6%, and 42.4%, respectively. As with the increase in age, the disability rates of ADL, IADL, PADL, and their 14 items gradually increased (p < 0.05), with walking, using the telephone, and using public vehicles having higher disability rates than other items. The influencing factors of ADL disability were gender , age , educational level , and having metabolic syndrome (MetS) . Compared with ADL, the influencing factor of PADL disability was gender, whereas that of IADL disability was whether someone had MetS. Conclusions: With age, the possibility of ADL, PADL, and IADL damage in the elderly is higher. Gender, age, educational level, the number of chronic diseases, and whether someone has MetS might be the influencing factors of disability. Interventions should be taken from a variety of sources specific to the content of each entry. By the end of August 2015, the Geriatric Society of China announced that Guangxi was the largest province in China with a population exhibiting good longevity rates ,4. HowevAccording to estimates, by 2050, the population of people aged 80 around the world would reach 400 million, 70% of whom can be found in the underdeveloped regions . Recent With the development of the ageing population, the health condition of the elderly and the major influencing factors have attracted significant scholarly attention. Dealing with the risks and burdens of disability caused by ageing is crucial. The ability of daily life (ADL) refers to the most basic and common body movements that people must perform repeatedly every day to live independently . ADL conFrom June 2016 to July 2017, we recruited 2300 elderly people aged 60 and above from three townships in a County . Of all the towns in the County, according to the proportion of the aged over 60 in the total population of the town, we chose the top three of the above proportion as the research site. All the older persons over 60 years of age in these three townships were the subjects of this study. The inclusion criteria were as follows: permanent residents with formal household registration and those who were aged 60 years or above at the time of the survey. The exclusion criteria are as follows: people with a mental system disease, Alzheimer’s, or severe cognitive impairment; those who refused investigation and physical examination; and those who lived beyond the areas being investigated.The project was initiated by obtaining data from the respondents aged 60 and above from the local Department of Health. The elderly were guided to the local hospital of the town under the care of village doctors and cadres. After obtaining consent, the investigators administered the questionnaire and conducted health examinations on the elderly. The investigators consisted of trained staff and physical examination doctors. If the elderly cannot complete the above tasks independently, family members assisted them in completing the relevant questionnaire survey and physical examination.The ADL Scale was adapted from the 1969 study of American scholars Lawton and Brody . The ADLMetabolic syndrome (MetS) is a group of risk factors for cardiac metabolic disorders including abdominal obesity, dyslipidemia, hypertension and hyperglycemia . AccordiData were recorded with the Epidata 3.1 database, and SPSS16.0 statistical software  was used for statistical analysis. The demographic characteristics of the elderly were described in terms of rate and percentage. The chi-square test was used to compare the disability rate among the elderly with different demographic characteristics. The graph is used to show the disability rates of ADL, six items of PADL, and eight items of IADL at different ages. To obtain the relationship between ADL, PADL, IADL and its 14 items and age, the trend chi-square test was used to analyze whether there was a trend between them. Binary logistic regression was used to analyze the influencing factors of disability rate in the elderly. The independent variables were gender, age, marital status, education level, number of chronic diseases, smoking or not, alcohol use, and whether they had MetS. The dependent variables were ADL, PADL, IADL, and 14 items (with/without disability). The results of the binary logistic regression analysis were expressed in OR with a confidence interval of 95%. The test level was 0.05 on both sides.A total of 2300 elderly people agreed to participate in this study. The sample had the following characteristics: 58.7% females (1350 out of 2300); the youngest and oldest were 60 and 104 years old, respectively (average age: 70.32 ± 7.93 years), those aged 60~ years accounted for 53.2% (1350 out of 2300), 70~ years 37.2% (855 out of 2300), 80~ years 5.0% (116 out of 2300), and 90 years old and above accounted for 4.6% (106 out of 2300). The ethnic group is dominated by the Zhuang people, accounting for 81.3% (1869 out of 2300) of the sample. The majority of the sample was illiterate, accounting for 41.3% (1038 out of 2300). Those who were married accounted for 68.0% (1564 out of 2300). Those who were widowers accounted for 8.8% (203 out of 2300), widows accounted for 23.1% (532 out of 2300). Most of the elderly comprised farmers, accounting for 93.3% (2147 out of 2300), while the non-smoking and non-drinking respondents accounted for 83.3% (1917 out of 2300) and 76.7% (1763 out of 2300) of the sample, respectively .p < 0.05). The disability rate of females was higher than that of males. With age, the elderly had a higher disability rate. The disability rate in marriage was lower than that in non-marriage. As the level of education declined, the disability rate of elderly gradually increased. The incidence of elderly with smoking, drinking, and MetS was higher than that of those without smoking, drinking, and MetS.The overall disability rate of ADL was 43.4% (998 out of 2300). A statistically significant difference was observed in ADL disability rates among the following variables: gender, age, occupation, marital status, educational level, whether or not the person was smoking and drinking, and whether the person had MetS (p < 0.05). The disability rate in marriage was lower than that in non-marriage, and the disability rate was higher with the decline of education. In addition, as the number of chronic diseases increased, the disability rate increased gradually. Finally, the disability rate of the elderly with MetS was higher than that without MetS.The disability rate of PADL was 11.6% (266 out of 2300). Statistically significant differences were observed in the PADL disability rates among age, marital status, number of chronic disease, and whether with MetS (p < 0.05) (The disability rate of IADL was 42.4% (976 out of 2300). The disability rate among different characteristics was the same as that of ADL. Except for ethnic groups and the number of chronic disease, the disability rates among other features were statistically significant ( < 0.05) .p < 0.05)  . As reve < 0.05) . Their d < 0.05) .As shown in The number of chronic diseases might be an influence factor for walking , bathing , toileting , preparing a meal , performing household tasks , taking medication , doing some washing , and grocery shopping  . The presence of MetS in an individual might be an influencing factor for walking , grooming , toileting , doing financial management , preparing a meal , performing household tasks , doing some washing , and grocery shopping   As the aging population continues to increase, maintaining the functional capacity of older adults has become an urgent concern . The occThe IADL disability rate in the present study is higher than Shimada’s findings 26.7%) for 10,885 community-dwelling older adults aged 65 and enrolled in the NCGG-SGS . The tre.7% for 1Logistic regression analysis results showed that gender may be the influencing factor of ADL and IADL disability in the elderly, as females were more likely to encounter ADL and IADL disabilities than males. This finding is consistent with the results of past studies ,29,30. AThe trend chi-square test showed that the rate of disability increased with age. Logistic regression analysis results also indicated that age was the influencing factor for disability, as all the results  showed that the older the age was, the higher the OR value was. This finding is consistent with many studies ,32,33,34Meanwhile, educational level also served as an influencing factor of ADL and IADL. Five of the eight items of IADL showed that the lower the education level was, the higher the incidence of disability. Scholars believe that the high frequency of ADL disability is related to low education level ,38,39,40In this study, although the results of multiple factors failed to conclude that alcohol consumption was not a possible influencing factor for the occurrence of ADL, PADL and IADL, it was concluded that alcohol consumption could be influencing factor for seven items. From the OR value, someone with alcohol use were less likely to occur disability than someone without alcohol use. Human activity and cognitive function were affected by alcohol use . StudiesStudies have shown that the number of chronic diseases is associated with impaired ADL, and the more chronic diseases there are, the higher the rate of ADL disability would be ,47,48. EStudies have also found that MetS is significantly related to the ADL and IADL of the elderly living in the community ,54,55. HThis study has the following highlights. To the best of our knowledge, this is the first study to analyze the influencing factors of disability among the elderly in the county . Second, many current studies have analyzed the disability of the elderly but only by analyzing the overall ADL, PADL, IADL and few have studied each item in greater detail. In comparison, this study analyzed the risk factors for disability from ADL, PADL, IADL and each activity (14 items) to explore the possible factors of disability among the elderly living in the mountain areas. The study provides a scientific basis by offering basic data for further interventions, which can allow the local elderly to have a better quality of life. Third, for certain influencing factors, although multivariate analysis failed to show certain factors were risk factors for ADL, PADL, or IADL disability, a risk factor was indicated in certain specific activities (14 items reflected).Several aspects, however, need further attention. First, as this survey is a cross-sectional study, it cannot determine the causal relationship between influencing factors and disability. Second, as the data were self-reported, individual bias may have influenced the information. Third, the number of samples in this study was not large enough. For this reason, enlarging the number of samples is needed to increase the credibility and extensibility of the results. This study is the baseline survey of our project, and we aim to do a follow-up study on the elderly who have participated in the survey every three years in the future.Among the elderly in a County in Guangxi Province, China, an increase of age also increases the likelihood of being disability in terms of ADL, PADL, and IADL. Gender, age, educational level, the number of chronic diseases, and whether someone has MetS might be the influencing factors of disability. For some functions of daily life, alcohol use might play a role in relieving the decline in functional, further researches should be made to confirm it.


Hearing loss is a prevalent sensorineural disorder and a major public health issue in China. It is suggested that half of all cases of hearing loss can be prevented through public health measures. However, national strategies for hearing healthcare are not implemented well in Guangdong and some other regions in China.To develop a community-based service model for the prevention and control of hearing loss in Guangdong, we integrated the model with multiple maternal and child healthcare models, and set up a series of clinical programs along with an optimum timeline for the preventive measures and intervention treatments to take place. A total of 36,090 families were enrolled in the study, including 358 high-risk families and 35,732 general-risk families.The study lasted for 6.5 years, and 30,769 children were born during that period. A total of 42 children were born with congenital deafness; 17 of them were born into families with advanced genetic risks for hearing loss, 9 were born with specific medical conditions, and 16 were born into general-risk families. About one third of them were diagnosed prenatally, others were diagnosed within 3 months of age, and 72% of them received interventions initiated before 6 months of age. 13 children presented with delayed hearing loss; 9 of them were diagnosed with delayed hereditary sensorineural deafness in neonatal period, and 4 were diagnosed within 3 months after onset. Timely interventions were provided to them, with appropriate referrals and follow-ups. Beside these, 80 families were identified with genetic susceptibility to aminoglycoside ototoxicity. Detailed medication guides were provided to prevent aminoglycoside-induced hearing loss. Moreover, through health education and risk reduction strategies, the prevalence of TORCH syndrome decreased from 10.7 to 5.2 per 10,000. Additionaly, the awareness rates of health knowledge about hearing healthcare significantly increased in the cohort.Adapting national strategies for local or district projects could be an important step in implementing hearing loss prevention measures, and developing community-based service models could be of importance in carrying them out. Hearing loss is a prevalent sensorineural disorder and a major public health issue, associated with increased cognitive obstacles, poorer social functioning, and increased health care costs –3. AccorHearing loss may result from genetic factors, infection factors, perinatal or neonatal complications, ototoxic drugs, excessive noise and other causes . It is sThe study was approved by Ethics Committee of Guangdong Women and Children Hospital of Guangzhou Medical University. A total of 36,090 families were enrolled in the study under an institutional review board-approved protocol. The cohort included 358 high-risk families (with hearing impairments or family histories of hearing loss), as well as 35,732 general-risk families . The study cohort was recruited from October 2010 to February 2017. All participants have given their written informed consents. All the procedures performed in the study were in accordance with the Declaration of Helsinki.During pre-conception counseling, medical records for each participating families were obtained, including clinical histories, infection histories, histories of abnormal pregnancies, use of ototoxic drugs, occupational exposures, as well as genetic factors related to hearing loss. For families with hearing impairments or family histories of hearing loss, clinical evaluation and genetic testing were recommended. As to hearing families without family history of hearing loss, health education and screening tests were provided. For couples identified with pathogenic mutations, recurrence information and reproductive choices were provided. Moreover, TORCH  complex screening was recommended to pre-pregnant women for the prevention and control of toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus, HIV, syphilis and other common congenital infections. Effective vaccines are available for many of the viruses that cause hearing loss, and may lead to substantial changes in the incidence of these infections in the offsprings of these pre-pregnant women. Interventions for the prevention and treatment were undertaken according to the guidelines . AdditioClinical evaluation and genetic testing were provided to 358 high-risk families through the diagnosis program of hearing loss. Physical examination and screening tests were provided to 35,732 general-risk families through a screening program. In the screening program of hearing loss, allele-specific PCR-based universal array was applied to screen hotspot mutation carriers of deafness-associated genes. In the diagnosis program, a microarray assay was used to initially select hotspot mutations of genes associated with hereditary hearing loss, and for those with hearing impairment but tested negative for hotspot mutations, DNA sequence analysis were performed. Details about both the programs were described in our previous reports , 15. TheImprove birth practice, and try to avoid birth trauma and hypoxia. For high-risk participants with HSV lesion or prodrome presented at the time of delivery, cesarean section was recommended according to guidelines to avoid vertically transmitted infection . For neoUniversal newborn hearing screening (UNHS) was carried out for infants, using the transient evoked otoacoustic emissions and auditory evoked potentials as the first two steps, and automated auditory brainstem response (ABR) test as the third step. As to cases with increased genetic risks, genetic testing was also recommended. Moreover, we screened for G6PD deficiency, blood group incompatibility and other risk factors. In those newborns with advanced risks of hemolytic diseases, timely measures were taken to avoid jaundice caused auditory neuropathy and hearing loss. In addition, avoidance of ototoxic drugs and reduction in exposure to excessive noise were recommended for the prevention of hearing impairment.In childhood and adolescence, health education is important in promoting personal hygiene and avoiding impacted cerumen, as well as in preventing otitis externa. Better management of upper respiratory tract infections help reduce otitis media. Immunization and prophylaxis help avoid measles, mumps and meningitis. Thus, for children and adolescents, we developed a student hearing healthcare program to promote personal hygiene and ear care, to help them better manage upper respiratory tract infections, to promote immunization against illnesses related to hearing loss, and to increase their awareness about harmful factors. We gave health education in schools and communities, organized health knowledge quizzes and other fun activities to increase their awareness of harmful factors and preventive measures.To promote a positive image of hearing, we developed a family hearing healthcare program in our ear center and brought it to the communities. We gave health care consultations in the chinic, provided health education in the communities, hosted workshops and distributed brochures. Of those, excessive noise is a major avoidable cause of hearing impairment. In China, occupational noise and urban, environmental noise are significant risk factors for hearing impairment. Because there is widespread ignorance of the hazard, awareness should be increased about the harmful effects of noise on hearing and about the prevention and control of noise-induced hearing loss. Thus, in adults, protection against excessive and/or prolonged noise exposure, as well as ototoxic solvents and industrial chemicals were provided. Moreover, a surveillance system and a self-reporting system were developed in the program, so that timely interventions and appropriate referrals could be provided.In the present study, we integrated the service model for the prevention and control of hearing loss with multiple maternal and child healthcare models, and developed a community-based service model to prevent hearing loss from major causes. A series of clinical programs along with an optimum timeline Fig.  were adoA total of 126 couples were identified with increased genetic risks for hearing loss. After genetic counseling, 3 carrier couples chose to receive pre-implantation genetic diagnosis, 96 high-risk couples decided to proceed prenatal diagnosis, while another 27 couples chose to take audiological evaluation and genetic testing postnatally. Of them, 11 deaf couples carried biallelic mutations on the same genes would inevitablely have children with autosomal recessive hearing loss if they chose not to receive gamete donation. They were provided with detailed information and early intervention programs. Moreover, TORCH complex screening was carried out during (pre)pregnancy examination. The prevalence of serum antibodies to TORCH pathogens in the study cohort was as below. The specific IgM antibodies were found to be positive in 2.68% cases for CMV, 1.04% cases for toxoplasmosis, 1.07% cases for the Rubella virus and 0.45% for the HSV infections. The seropositvity for anti-CMV IgG (+) was 97.35%, anti-Toxoplasma IgG (+) was 25.37%, anti-HSV IgG (+) was 21.54% and anti-Rubella IgG (+) was 83.87%. Interventions for the prevention and treatment were undertaken according to the guidelines [To prevent TORCH syndrome, appropriate treatments and risk reduction strategies were employed in perinatal stage. For 7 high-risk participants with HSV lesion or prodrome presented at the time of delivery, cesarean section was recommended according to guidelines  to avoidA total of 24,746 newborns received UNHS, 1739 failed the initial hearing screening, and 193 of them failed the secondary screening. Those who failed both hearing screening were given audiological assessment at their third months, and 16 were diagnosed with hearing impairment. Of those, 8 were with mild hearing impairment, 6 were moderate hearing impairment and 2 were severe hearing loss. In the case of another 6023 newborns with advanced genetic risks or specific medical conditions, audiological assessment and genetic testing were provided, which identified 26 with hearing loss. Intervention programs were provided to them, and 72% received appropriate interventions initiated before 6 months of age, which was significantly ahead of the intervention time before (around 2 years old). Subsequent hearing rehabilitation through hearing aids and cochlear implants as well as suitable therapy were provided in the program. Moreover, 9 infants were indentified with deafness-associated mutations and may develop into delayed hereditary sensorineural deafness, another 7 might have genetic susceptibility to aminoglycoside ototoxicity. Detailed medication guides and timely intervention were provided to their families, with appropriate referrals and follow-ups.A student hearing healthcare program was developed for children and adolescents to promote personal hygiene and ear care, to promote immunization against illnesses related to hearing loss, and to increase their awareness about harmful factors. We gave health education in schools and communities, to promote a positive image of hearing in children and adolescents. According to questionnaire surveys before and after health education, the awareness rate of principal health knowledge about hearing healthcare increased from 37 to 86%.To get across common causes and preventive measures of hearing impairment, we provided health education through a family health care program and an occupational disease prevention program. During the study period, we gave health care consultations to more than 3000 families, provided 12 times of health education in the communities, hosted workshops for 11 times and distributed more than 8000 brochures to increase public awareness about harmful factors related to hearing impairment. According to the questionnaire survey, the awareness rate of common causes and preventive measures of hearing impairment increased to 78% in the adult respondents after the study. According to our survey, excessive noise is a major avoidable cause of hearing impairment. Thus, in adults, protection against excessive and/or prolonged noise exposure were provided. Besides, improper use of aminoglycoside antibiotics is a comparatively common cause of hearing loss in China . Since mTo develop a community-based service model for the prevention and control of hearing loss, a total of 36,090 families were enrolled in the study, including 358 families with hearing impairments or family histories of hearing loss, and 35,732 hearing families without family history of hearing loss. The study lasted for 6.5 years, and 30,769 children were born during that period. A total of 42 children were born with congenital deafness, 17 of them were born into families with advanced genetic risks for hearing loss, 9 were born with specific medical conditions like prematurity, low birth weight, hyperbilirubinaemia and NICU stay, another 16 were born into general-risk families. About one third of them were diagnosed prenatally, others were diagnosed within 3 months of age, and 72% of them received interventions initiated before 6 months of age. Besides, 13 children presented with delayed hearing loss; 9 of them were diagnosed with delayed hereditary sensorineural deafness in neonatal period, and 4 were diagnosed within 3 months after onset. 1 adolescent and 5 adults suffered from hearing impairment because of accidental injuries, 2 adolescents and 7 adults appeared tinnitus, and were all diagnosed within 3 months after onset. They were detected in the ongoing surveillance system and also the self-reporting system in the program, and were provided with timely interventions as well as appropriate referrals.Moreover, 80 families were identified with mutations in mitochondrial DNA 12S rRNA gene, which are important mechanisms of genetic susceptibility to aminoglycoside ototoxicity , 20. GivFurthermore, the awareness rates of principal health knowledge about hearing healthcare significantly increased through health education. In (pre)pregnant families, the awareness rate increased from 42 to 90% after receiving (pre)pregnancy counseling services. For children and adolescents, the health education have been brought to schools, and the awareness rate increased from 37 to 86%. For adults, health education were provided through occupational disease prevention system, and the awareness rate increased to 78% after the study.It is suggested that half of all causes of hearing loss can be prevented through public health measures . EspeciaThe service model we developed was integrated with multiple maternal and child healthcare models, including preconception care models, prenatal care and fetal monitoring models, perinatal health care models, neonatal screening models, student healthcare programs, and occupational protection programs. So that the service model for the prevention and control of hearing loss was no longer isolated or periodical, and became integrated and continuous. Given that the government input in the prevention of hearing loss was limited and the medical insurance was not satisfying, the national strategies for hearing healthcare are not implemented well in Guangdong and some other regions in China. Whereas, the maternal and child healthcare has solid foundation in China, and the service models are inclusive. Hence, we try to integrate the service model for the prevention and control of hearing loss with multiple maternal and child healthcare models, and it demonstrates with favorable performance in the clinical practice of Guangdong Province.As important components of the model, we provided primary ear and hearing care to prevent and treat common ear conditions , 23, carWith the world’s largest population and the new family-planning policies, hearing loss has become a significant public health concern in China. Developing community-based service models could be of importance in implementing national strategies to prevent and control hearing loss. The service model we developed was integrated with multiple maternal and child healthcare models, and demonstrated with favorable performance in the clinical practice of Guangdong Province.


Rhizopusarrhizus is the most common agent isolated worldwide, Apophysomycesvariabilis is predominant in Asia and Lichtheimia species in Europe. The new causative agents, Rhizopus homothallicus, Mucor irregularis, and Thamnostylum lucknowense are reported from Asia. In conclusion, with the change in epidemiology of mucormycosis country-wise studies are warranted to estimate disease burden in different risk groups, analyse the clinical disease pattern and identify the new etiological agents.Mucormycosis is an angio-invasive fungal infection, associated with high morbidity and mortality. A change in the epidemiology of mucormycosis has been observed in recent years with the rise in incidence, new causative agents and susceptible population. The rise has been perceived globally, but it is very high in the Asian continent. Though diabetes mellitus overshadow all other risk factors in Asia, post-tuberculosis and chronic renal failure have emerged as new risk groups. The rhino-cerebral form of mucormycosis is most commonly seen in patients with diabetes mellitus, whereas, pulmonary mucormycosis in patients with haematological malignancy and transplant recipients. In immunocompetent hosts, cutaneous mucormycosis is commonly seen following trauma. The intriguing clinical entity, isolated renal mucormycosis in immunocompetent patients is only reported from China and India. A new clinical entity, indolent mucormycosis in nasal sinuses, is recently recognized. The causative agents of mucormycosis vary across different geographic locations. Though  Mucorales. Humans acquire the infection predominantly by inhalation of sporangiospores, occasionally by ingestion of contaminated food or traumatic inoculation [Mucorales are ubiquitous, and morphologically appear as broad, aseptate or sparsely septate ribbon-like hyphae. Eleven genera and ~27 species under Mucorales are associated with human infections. Rhizopus arrhizus is the most common agent causing mucormycosis across the globe, followed by Lichtheimia, Apophysomyces, Rhizomucor, Mucor and Cunninghamella species [Mucormycosis is caused by the fungi belonging to the order culation ,2. The f species ,4,5.Mucormycosis is associated with angio-invasion and high mortality ,5. The iThe incidence of mucormycosis is rising globally ,13,14,15The population-based prevalence of mucormycosis is reported from a few countries. In San Francisco, California, a population-based surveillance study conducted during 1992 to 1993 documented the cumulative annual prevalence of invasive fungal infections at 178.3 per million, with mucormycosis at 1.73 cases per million . An autoThe true incidence/prevalence may be more in mucormycosis, as many of the cases remain undiagnosed due to difficulty in collecting the sample from deep tissue and low sensitivity of diagnostic tests. The Leading International Fungal Education (LIFE) portal has estimated the burden of serious fungal infections globally. According to their estimate, the annual prevalence of mucormycosis might be around 10,000 cases in the world barring India. After the inclusion of Indian data, the estimate of mucormycosis rose to 910,000 cases globally ,24. The The common underlying diseases in mucormycosis include diabetes mellitus with or without diabetic ketoacidosis, malignancies , transplant recipients (haematopoietic stem cell and solid organ transplants), corticosteroid therapy and neutropenia. However a considerable number of cases have been reported in patients without any underlying disease or risk factors. A contrasting difference in underlying disease/risk factors has been noted between the western world (USA and Europe) and the Asian region. Diabetes mellitus is the most common risk factor associated with mucormycosis in India ,16,17, cDiabetes mellitus as predisposing factor varies from 17% to 88% globally ,65,66. TThe prevalence of diabetic population is estimated to rise from 366 million in 2011 to 522 million in 2030 globally . China tHaematological malignancy (HM) was the most common underlying disease for mucormycosis in Europe and the United States, ranging from 38% to 62% . PatientSolid organ malignancies and solid organ transplant (SOT) recipients are also important risk factors for mucormycosis though in a small proportion of cases . VariousRhizopus species and iron helps in the growth of those fungi. An international registry on dialysis patients registered 59 cases of mucormycosis and 70% of those patients had no known underlying disease. However, 78% of the patients had deferoxamine therapy, which made the authors suspicious about deferoxamine therapy as a risk factor for mucormycosis [Iron overload and deferoxamine therapy play a major role in the pathogenesis of mucormycosis. Earlier deferoxamine was commonly used to reduce iron/aluminium overload in patients with diabetic ketoacidosis (DKA), haemodialysis, renal failure and transfusion related disorders . The irormycosis . The morrmycosis ,87. Howermycosis ,89. An ormycosis . Howeverrmycosis . A recenLionakis et al. reviewed breakthrough invasive mould infection after antifungal prophylaxis in haematological malignancy and HSCT patients . The stuOther risk factors associated with mucormycosis are HIV infection, intravenous drug use, low birth weight infants, malnutrition, chronic alcoholism, liver diseases, chemotherapy and use of calcineurin inhibitors ,5. A recMucormycosis is not only the disease of the immunocompromised host; a considerable number of cases are seen in an immunocompetent host without any known underlying illness. Two meta-analysis at different time periods documented ~19% of the mucormycosis cases occur in immunocompetent hosts ,4. ThoseRhizopus species was isolated from 42% of the samples collected from clean linens [Though mucormycosis was considered a community-acquired disease, nosocomial mucormycosis has been increasingly reported from many hospitals ,113. Docn linens . Since iBased on the anatomical site of involvement, mucormycosis is classified into rhino-orbito-cerebral (ROCM), pulmonary, gastrointestinal, cutaneous, renal, disseminated and other miscellaneous forms, which include infection of bones, heart, ear, parotid gland, uterus, urinary bladder and lymph nodes ,4. The cROCM is the most common form and it is often seen in patients with diabetic ketoacidosis or with uncontrolled diabetes mellitus ,4. A stuThe imaging studies may help to delineate the extent of tissue invasion. The computed tomography and magnetic resonance of paranasal sinus mucormycosis included mucosal thickening, osseous erosion, sinusitis with hypo/mild/hyper intense lesions, and destruction of bones in nasal septa, orbit, maxilla and mandible. In case of orbital and cerebral extension, the disease can be seen as orbital cellulitis, optic neuritis, soft tissue infiltration in the optical apex, bone rarefaction and erosion of the skull base, cavernous sinus and internal carotid artery thrombosis, infracts and intra-cranial abscess in the brain ,128.Pulmonary type is the second most common site of involvement and often seen in patients with haematological disorders and transplant recipients ,5. HaemaCutaneous mucormycosis often occurs after trauma or breach of skin and can be seen in the immunocompetent host. A review on cutaneous mucormycosis reported that 43–67% of the patients were apparently immunocompetent . DiabeteGastrointestinal (GI) mucormycosis is the most difficult disease to diagnose ante-mortem, and commonly seen in low birth weight infants, in patients with malnutrition or undergoing peritoneal dialysis ,135. AmoVarious studies from India documented the rise in patients with isolated renal mucormycosis from 5.4% to 14% among all mucormycosis cases ,16,17. IMucorales are angio-invasive, and can disseminate through haematogenous route. A meta-analysis reported 13% of the mucormycosis cases present with disseminated disease. Lung (91.2%) is the most common site of dissemination followed by the central nervous system (53%), sinus (32.4%), liver (17.6%) and kidney (14.7%) [As  14.7%) . Solid o.7% . SolMucorales are known to cause mucormycosis [Mucorales in various studies across the globe. Species under Genera Rhizopus, Lichtheimia, and Mucor are common causative agents and Rhizopus arrhizus is the most common agent [Rhizopus species was often associated with ROCM form of the disease; Cunninghamella species with pulmonary disease or disseminated disease. Whereas, Apophysomyces and Saksenaea species were commonly isolated from cutaneous mucormycosis [Rhizopus species and not Lichtheimia, where as corticosteroid predisposes Lichtheimia species [Cunninghamella species was found be significantly higher than infection with any other species under Mucorales [Lichtheimia species are common in Europe, it is relatively rare in other regions [Apophysomyces variabilis is the second most common cause of mucormycosis in India [Apophysomyces species [Apophysomyces variabilis, infections due to R. microsporus and R. homothallicus are on the rise in India [R. homothallicus was isolated from patients with ROCM type, pulmonary and cutaneous form of disease [R. homothallicus was reported from France [Mucorales such as Cokeromyces recurvatus, Syncephalastrum species, and Saksenaea species are rarely reported [Mucor irregularis infection was reported from China and India [Mucor velutinosus and Mucor ellipsoideus have been reported from the USA [Thamnostylum lucknowense was reported from India [Mucorales are thermo-tolerant fungi, ubiquitous in the nature, and are widely found in organic decaying materials [Mucorales species in soil [Apophysomyces variabilis and Rhizopus homothallicus are found in India soils. Eleven genus and ~27 species under the order rmycosis ,4,139. T species . Mortali regions ,11,70. Ain India . India a species ,140,141.in India ,142. R.  disease ,143,144.m France . Infectind India ,147. Inf the USA . A case om India . Mucoral in soil . The so Rhizopus homothallicus, Thamnostylum lucknowense, and Mucor irregularis are the new emerging species. It warrants population-based studies on specific risk population  to understand global epidemiology of the disease.The exact burden of mucormycosis is not known, as it is not a reportable disease and rare in developed countries. The disease is common in developing countries, but the laboratory facility is sub-optimal in this region. The estimate of mucormycosis has been accessed from large series, though majority studies did not have any denominator. Few population-based studies estimated the incidence of mucormycosis in the western world, but the same does not represent the picture in developing countries, as the risk groups and interventions are different between these two worlds. In developing countries, the incidence of diabetes mellitus is alarmingly high, which may lead to a rise in the incidence of mucormycosis. In developed countries, the rise in incidence of mucormycosis is linked to intense immunosuppression in haematological malignancies and transplant recipients. A change in epidemiology of mucormycosis has been noted in recent years with emergence of new risk factors and causative agents. Post-tuberculosis, chronic renal failure and stay in intensive care unit are new risk factors for the disease especially in developing countries.