H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans. Highly pathogenic H5N1, human H7N9, and more recently H10N8 possess the internal gene cassette from poultry H9N2 viruses; thus, it has been hypothesized that poultry H9N2 virus, in addition to retaining the ability to infect humans, might enable H5, H7 and H10 subtypes to infect and transmit to humans.Influenza is a serious ongoing threat to human and animal health. Every year, thousands of people are infected with seasonal influenza. In addition, they may be exposed to avian  and swine (H1 and H3) influenza subtypes.8 Distinguishable lineages of H9N2 have become enzootic in land-based poultry in Asia, Africa and the Middle East.12 In poultry, H9N2 infection is usually asymptomatic, with little to no mortality and decreased egg production.13 Experimentally, H9N2 virus infects mice and has been isolated from pigs and dogs in China.16 H9N2 has also sporadically infected humans without causing mortality. To date, 11 human H9N2 infections have been reported in China; two cases have been reported in Bangladesh and three in Egypt.19 Recent phylogenetic classification of the H9N2 shows that this subtype has evolved into 23 distinct clades with five clade-specific outliers.20 The G1-like lineage represented by A/quail/Hong Kong/G1/1997 and the Y280-like lineage represented by A/chicken/Hong Kong/G9/1997 are the two most predominant progenitor lineages of the H9N2 viruses currently circulating in China, the Middle East and Southeast Asia.22 Surveillance reports from the Middle East, India and Pakistan, show that H9N2 viruses circulating in those areas are genetically related to the G1 clade but have evolved into distinct subclades.23In North America, the H9N2 subtype was first isolated from turkeys in 1966.25 Bangladeshi H9N2 viruses can be traced back to the G1 lineage.23 Since their introduction during the early 2000s, these H9N2 viruses have evolved tremendously via intra- and intersubtype reassortment and genetic drift to become unique and distinct from the prototype G1 viruses.23 Bangladeshi H9N2 avian influenza viruses' hemagglutinin (HA) belongs to the Iranian H9N2 lineage (Mideast_B); its neuraminidase (NA) and polymerase basic 2 (PB2) genes are from the Dubai H9N2 lineage (Mideast_C Dubai); and the nonstructured protein (NS), nucleocapsid protein (NP), matrix (M), polymerase acidic (PA) and polymerase PB1 genes are from highly pathogenic avian influenza (HPAI) H7N3 viruses originating in Pakistan.23 In addition, Bangladeshi H9N2 viruses have acquired numerous molecular makers throughout the genome that facilitate host-range transmission from avian species to humans.23Surveillance studies from Bangladesh have shown that H9N2 is endemic in chickens and quail and that live-bird markets serve as an epicenter for infection and transmission.ex vivo cultures of human cells and swine tissues and tested the viral susceptibility and transmission in mice and ferrets.Few studies have examined the replication potential and transmissibility of avian H9N2 viruses to mammals, and almost all of those have focused on G1 viruses isolated from China. To date, very little information is available on the pathogenesis, virulence and transmission of Bangladeshi H9N2 viruses. To close this gap, we determined the potential of Bangladeshi H9N2 viruses carrying H7N3 internal genes to replicate in mammals. We modeled the replication and pathogenesis of these viruses in All animal studies were approved by the St Jude Children's Research Hospital Animal Care and Use Committee and were performed in compliance with the policies of the National Institutes of Health and the Animal Welfare Act. All experiments were carried out by trained personnel working in a United States department of agriculture–inspected animal biosafety level 3+ animal facility.50) according to the method of Reed and Muench.26The H9N2 viruses used in this study  were isoL-glutamine (2 mM), 5% fetal bovine serum and antibiotics at 37 °C with 5% CO2. Normal human bronchial epithelial (NHBE) cells  from two healthy male donors (two and four years old) were expanded, cryopreserved and maintained in culture in an air/liquid interface (ALI) system, as previously described.28 Briefly, cells were plated in 0.33 cm2 transwell inserts  and allowed to differentiate. An ALI was established when the cells reached 98%–100% confluence. Media from the apical surface of the cells was removed, and the cells were exposed to a humidified 95% air/5% CO2 environment. The basolateral media or the ALI media, which was supplemented with SingleQuot growth factors (Lonza), was changed every 48 h for a minimum of six weeks. During every media change, the apical surface of the cells was washed with sterile phosphate-buffered saline (PBS) to remove mucus.Madin-Darby canine kidney (MDCK) cells  were serially passaged and maintained in culture in modified Eagle's medium  supplemented with 30 Briefly, the isolated tracheas were washed with PBS containing penicillin–streptomycin–amphotericin B , split open lengthwise and pinned to a dissection board. Intact tracheal biopsy explants were obtained using a disposable 5-mm biopsy punch. The isolated explants were then incubated at 37 °C in 5% CO2 at an ALI in transwell inserts similar to conditions used to grow the NHBE cells for at least 24 h before infection, with the following exception: the basolateral medium was changed hourly for the first 4 h.The replication properties of the Bangladeshi H9N2 viruses were tested in juvenile swine tracheal and lung explants. Juvenile trachea and lung were obtained from female piglets . Due to logistical limitation and availability of juvenile respiratory tissue, we conducted only one independent experiment in duplicates. Both trachea and lungs were harvested within 1 h of killing and processed as described.et al.29 The isolated lungs were first perfused with cold transport inserted through the bronchiole, perfused again with 1% low-melt agarose in PBS (Sigma), surrounded with icepacks until the agar solidified. The agarose-perfused lungs were then dissected into smaller tissue pieces (~2 × 1 × 1 cm size) and embedded in 4% agarose. Thin slices were prepared from embedded lungs with a 1-mm microtome blade and explants (5 mm diameter) were prepared with biopsy punches handled similarly to the tracheal explants.For the lung tissue, we adopted and modified the method described by Chan 6 EID50 units per explant in 100 μL of infection media was prepared for each virus and added to the apical surface of the tissues. Viruses were allowed to adsorb at 37 °C; after 1 h, the virus dilutions were removed by aspiration, and the apical surface was washed with PBS (pH 2.2) × 3 and PBS (pH7.0) × 3 to remove unbound virus and reduce background. Tissues were transferred to a new well containing fresh ALI medium with 1 × antibiotic/antimycotic and incubated at 37 °C. Samples were collected from the apical surface at 28, 48, 72 and 96 h. At the indicated time point, 300 μL infection medium was added to the apical surface for 30 min at 37 °C and then removed. Viral titers were determined immediately on MDCKs, with 50% tissue culture infectious dose (TCID50) determined using the method of Reed and Muench.26 Both the NHBE cell and swine-explant data are representative of combined data from two independent tests of three inserts per virus group.Prior to H9N2 infection, the apical surfaces of the NHBE cells or explants were washed with PBS pre-warmed to 37 °C, followed by equilibration with bronchial epithelial cell basal medium (Lonza) infection medium containing 0.5% bovine serum albumin at 37 °C for 30 min. Multiplicity of infection of 0.01 for NHBE cells or 106 EID50 units of virus in a 30-μL volume. Mice (n=5/virus group) were monitored daily for morbidity  and mortality. Two mice were killed at three and five days post infection (d.p.i.), respectively, and a section of lung was removed and homogenized in 1 mL PBS. Viral titer (TCID50) values were determined as previously described. At 14 d.p.i., blood from each of the experimental groups was collected and subjected to the hemagglutination inhibition (HI) assay31 with chicken red blood cells and homologous H9N2 viruses to test for seroconversion.Six-week-old Balb/c mice  were lightly anesthetized with isoflurane  and intranasally administered 10n=3 for each virus group) were anesthetized using isoflurane, and 106 EID50 units of avian influenza virus in 1 mL solution were administrated intranasally. Twenty-four hours later, direct contacts (n=3 for each virus group) and aerosol contacts (n=3 for each virus group) were introduced into their respective cages. Every 24 h, all ferrets' temperatures and weights were recorded. At 2, 4, 6, 8 and 10 d.p.i., the ferrets were sedated with ketamine (25 mg/kg body weight),33 and nasal washes were collected with 1 mL sterile PBS. The viral titers of the nasal washes were determined in MDCK cells, and TCID50 values were determined as previously described. At 16 d.p.i., blood was collected from all ferrets, and subjected to the HI assay31 with chicken red blood cells and homologous H9N2 viruses to test for seroconversion.3- to 6-month-old male ferrets , which were sero-negative for influenza A (H1 and H3 subtypes) and B, were used in the experiment. Two Bangladeshi H9N2 viruses were tested alongside the control H9N2 virus. For each virus group, three inoculated donors and three direct contacts were housed in the same cage, and three aerosol contacts were housed in an adjacent cage separated by a permeable barrier that allowed free airflow but not physical contact. Prior to infection, all of the ferrets were weighed, and their body temperatures were obtained. On the day of infection, donor ferrets (35 A/Fukui/20/2004 (E119) (H3N2) (wild-type virus) and A/Fukui/45/2004 (E119V) (H3N2) were used as reference viruses and are part of the World Health Organization's NAI-susceptibility panel.The NA inhibitors (NAIs) oseltamivir carboxylate , zanamivir  and peramivir  were used to test the sensitivity of the Bangladeshi H9N2 viruses. The NA activity was determined by a modified fluorometric assay using the fluorogenic substrate 2′-(4-methylumberlliferyl)-α-d-N-acetylneuraminic acid  as described previously.P-value ⩽0.05 was considered significant.Statistical analysis and graph were done using GraphPad Prism six software  and values were expressed as the mean±SD of the mean. A two-way analysis of variance test was used to determine the statistical significance for each individual H9N2 viruses that were used to infect NHBE cells. To determine the statistical significance between input inoculum and productive viral replication an analysis of variance comparison between 2 and 72 h post infection within the same virus was used. For both cell types a 25 and they are representative of the year, location and host species from which they were isolated. The nine H9N2 isolates were isolated during 2010–2013, and their genotypes are representative of H9N2 viruses that are circulating in chickens or quail. All of the viruses carry at least three internal genes  from HPAI subtype H7N3 from Pakistan.36 These selected viruses were screened for other influenza subtypes, including HPAI H5N1, and for Newcastle disease virus. One isolate, A/quail/Bangladesh/19462/13, was a H9N2/H5N1 mixture, and the H9N2 was isolated from the mixture by two simultaneous egg-neutralization passages in the presence of H5N1 antisera. The absence of HPAI H5N1 was confirmed using H5-specific real-time probes and conventional PCR primers.To assess the biologic properties of the Bangladeshi H9N2 viruses and their risk to mammals, we selected nine representative isolates  from our37 Six of the seven H9N2 isolates used in this study had the KSKR motif in the HA1/HA2 cleavage site, and this motif has been predominately identified in the Bangladeshi H9N2 isolates.25 In the NA, none of the isolates used in this study had both the E119V and R292K substitutions, which are associated with resistance to antiviral drugs such as oseltamivir and zanamivir. In the internal genes, numerous human host-specific changes were identified in PA, PB1, M, NP and NS (36 The substitutions were predominately in PB1 (L13P), NP (E372D), M1 (V15I), and M2 (L55F and S31N) and were associated with amantadine resistance  NHBE cells were sampled for virus replication, 2–72 h post infection (h.p.i.). All of the Bangladesh H9N2 viruses tested replicated in both the NHBE donors from a moderately low (2.5 Log10 TCID50/mL) to high titers (7 Log10 TCID50/mL) with a detectable difference in replication kinetics between the chicken and quail viruses as well as between the donors. None of the Bangladeshi viruses showed qualitative cytopathic effect in the NHBE cells. In both the donors cells within 24 h.p.i., the chicken H9N2 viruses with the exception of env/Bangladesh/8202 in NHBE donor 1 had viral titers (1–3.8 log10 TCID50/mL) similar to or higher than the mammalian control viruses (A/swine/Missouri/ 2124514/2006 (H2N3) and A/Ca/04/2009 (H1N1) virus ; 10 TCID50/mL at 48 h.p.i. to 6.5 log10 TCID50/mL at 72 h.p.i., (10 TCID50/mL) until after 48 h.p.i.  of the seven H9N2 viruses tested in trachea. Moderately low peak titer of 2.5 log10 TCID50/mL was mostly observed by 24 h.p.i. with no increase in viral titers till 72 h.p.i. (10 TCID50/mL) by 72 h.p.i. (10 TCID50/mL) at 48 h.p.i., whereas the negative control, A/Dk/NJ/872–27/78 (H2N2), had no detectable titers, after 72 h.p.i. (10 TCID50/mL by 48 h.p.i. Two of the three quail H9N2 viruses (env/Bd/10306 and env Bd/17403) showed delayed replication kinetics where viral titers were only observed only after 48 h.p.i. As expected, the positive control, A/swine/Missouri/2124514/ 2006 (H2N3), replicated to a high titer (6.3Log10 TCID50/mL) at 48 h.p.i., whereas the negative control, A/Dk/NJ/872-27/78 (H2N2), had no detectable titers, after 72 h.p.i. . Morbidity and mortality were monitored daily for 14 d.p.i. To determine the virus titer in the lungs, we infected a second group of BALB/c mice . Uninfected  and A/Puerto Rico/8/34 (n=5) (PR8)–inoculated mice served as controls. None of the BALB/c mice died or displayed clinical disease during the entire course of the study. As expected, control PR8-inoculated mice either lost more than 30% of their baseline body weight and were euthanized or were dead 4–5 d.p.i.  on both 3 and 5 d.p.i.  with differing molecular genotypes harboring various mammalian host-specific substitutions throughout their genomes, especially in the receptor-binding site of the HA glycoprotein . We infe10 TCID50/mL) than did the donor ferrets infected with env/Bd/10306 (~4 log10 TCID50/mL) at 2 d.p.i. (10 TCID50/mL), and the other ferret shed much smaller viral titers than did the donor (2.5–4 Log10 TCID50/mL) . Nasal titers from the donor ferrets from both H9N2 test groups showed that a productive infection was established within 2 d.p.i., and the animals were shedding virus nasally for at least 6 d.p.i.  Between 2 d.p.i. . Despite2 d.p.i. . In thatID50/mL) . No aeroID50/mL) . All ferID50/mL) . All ferID50/mL) . SeroconID50/mL) . All remID50/mL) .in vivo and in vitro. On the basis of this preliminary study, it appears that the Bangladeshi H9N2 viruses are capable of causing a productive subclinical infection in ferrets and might have the potential to transmit to animals in very close contact and under ideal conditions. However, none of the H9N2 viruses appeared to currently possess the potential for aerosol transmission.When the ferret nasal washes were compared with viral titers obtained from NHBE cells and swine explants, both the Bangladeshi H9N2 viruses had similar replication titers , and 6 i50 values ranging from 0.13 to 0.69 nM , as well as the NAI-susceptible human H1N1 and H3N2 influenza A viruses.46To test whether the Bangladeshi H9N2 viruses are sensitive to the currently approved antiviral drugs, we examined the susceptibility these H9N2 viruses to the NAIs oseltamivir, zanamivir and peramivir. All six H9N2 viruses tested were fully susceptible to the three NAIs, with mean IC 0.69 nM . These Iin vitro and in vivo mammalian model systems. We showed that the Bangladeshi H9N2 viruses can productively infect and replicate in NHBE cells, swine respiratory tissue, mice and ferrets after experimental inoculation. The H9N2 viruses replicated without causing any qualitative cytopathic effects in the in vitro model systems and replicated primarily in the respiratory tissue of mice and ferrets without inducing clinical signs of infection and were susceptible to currently approved NAIs. The lung appears to be the preferred site of replication for the H9N2 viruses.H9N2 avian influenza viruses are endemic in terrestrial poultry and have evolved into many permanent lineages  in Asia. The Bangladeshi H9N2 viruses used in this study belong to a distinct G1 sublineage containing multiple internal genes from HPAI H7N3 and harbor numerous mammalian host-specificity markers. Despite these numerous genotypic risk factors few studies have addressed the ability of these unique Bangladeshi H9N2 viruses to infect mammals. In this study, we investigated this issue by testing the viruses in various 47 but the higher replication titers that we observed were seen only after 48 h.p.i. Thus, the Bangladeshi H9N2 viruses can replicate in NHBE cells, despite lesser prevalence of the α-2,3 sialic acid linkages in the human cells.39 Studies have shown that H9N2 viruses harboring the Q226L mutation in HA display a 100-fold higher tropism for human airway epithelium when compared with H9N2 viruses with only the Q226 substitution.37 With the exception of the quail isolate (env/Bd/10306), all of the H9N2 isolates tested in this study had the Q226L mutation. In addition, all of the H9N2 viruses had the following mammalian host–specific mutations: HA , M1 (15I), M2 (55F and the drug-resistance marker 31N), NS (227K), NP  and PB1 (13P). This may explain why Bangladeshi H9N2 viruses may have recognized the α-2,6-linked sialic acid receptors present in the NHBE cells and were able to replicate in them. Studies of human H5N1 infection48 and swine-origin H1N1 infections50 have shown that molecular changes are required for adaptation to new host species.51 In addition, HA with modified glycans can recognize the α-2,3 sialic acid residue with decreased specificity and increased affinity,52 and a single amino acid substitution in the HA abrogates biding to the sialic acid residue.53 Therefore, understanding the molecular features of these avian H9N2 viruses is crucial to understanding their host-range switch.In the NHBE cells and swine tissue explants, the H9N2 viruses that we tested replicated to moderate-to-high titers. Previous studies in NHBE cells have shown that replication of influenza viruses between 8 and 24 h.p.i. directly correlates with infection in humans,41 Viruses that switch tropism from avian type to mammalian type also have tropism for the lower respiratory tract, as seen in the Bangladeshi H9N2 viruses.54 This result is also seen in α-2,3-binding H5N1 and α-2,6-binding H9N2 avian viruses.41 Alternatively host and viral process involved during an infection could influence the viral replication kinetics in pig trachea. Host defenses like the cell wall, mucosal surface and host factors that bind to virion components may prevent H9N2 viral replication. In addition, even if virus binds to the cell receptor, restriction to replication may also occur at different stages of the viral infection cycles. The host interferon responses during the initial stages of infection may protect the cell against influenza infection. The other factor that might influence replication in pig trachea is the genetic composition of the H9N2 viruses, that is, the current Bangladeshi H9N2 viruses may be poorly adapted and that mutations that are occurring within its genome are not optimized for adaption in the new host and may require further more compensatory mutation to attain replication competence in mammalian tissue. The preferential replication of the H9N2 virus in lungs, compared with that in the trachea, may be restrictive for these avian viruses to cross the species barrier. If the viruses are unable to establish a productive infection in the upper respiratory tract, then they are most likely to be cleared from the animal before they reach the lungs. This is consistent with the finding that H9N2 infections of man and swine are very sporadic.55 Therefore, unlike the other avian influenza subtypes like H1N1, H3N2, new reassortant viruses involving H9N2 viruses are less likely to use swine as the intermediate host/mixing vessel.Our swine explant data showed that H9N2 viruses replicate to less titers in juvenile swine tracheal cells, but the lung titers were similar to that of the swine control virus. Studies have shown that A/Quail/HK/G1/97 (H9N2), the progenitor of the Bangladeshi H9N2 viruses and the donor of the internal genes of HPAI H5 that infected humans in 1997, had similarly limited replication properties in the pig trachea compared to that in the lungs.In mice and ferrets, we found that the H9N2 viruses infect and replicate asymptomatically, as determined by lung and nasal titers. In the Balb/c mice, only one H9N2 virus (env/Bd/10306) caused weight loss between 3 and 5 d.p.i. without any other clinical signs. When the viral titers of lung samples were initially measured in MDCK cells, they showed very low titer, but when they were measured in eggs, the viral titer was comparable to other H9N2 viruses. This is because the env/Bd/10306 virus possesses the avian host-specific residue (Q226) in the HA to give a lower viral titer in MDCK cells than in eggs.56 have shown a delayed transmission pattern with H2N2 viruses that acquired the Q226L substitution in HA. Of the two H9N2 isolates tested (env/Bd/10306 and ck/BD/10450), only the chicken H9N2 virus carried the Q226L substitution. This supports the previous conclusion that Q226L substitution has an important role in mammalian transmission.37 The observed delay of direct-contact transmission and lack of aerosol transmission suggested that for the H9N2 avian influenza virus to adapt to humans, the virus may require one or many substitutions in addition to the Q226L substitution. Due to logistic limitations, we were unable to undertake a larger ferret study. However, the current results warrant a larger ferret study involving Bangladeshi H9N2 viruses with different molecular markers that have been identified previously.25 By doing this, we will be able to correlate and narrow the molecular determinates that are crucial for altering the host range of these viruses.The quail H9N2 virus (env/Bd/10306) and the chicken ck/BD/10450 virus replicated well in donor ferrets without inducing clinical signs. Two of the three direct-contact ferrets were infected with ck/BD/10450 virus after a delay of four days, and one of the direct contacts shed virus only at a single time point. Although not typically seen in ferret influenza transmission studies, two previous studies59 Several molecular determinants that influence receptor specificity and mammalian-tissue tropism have been identified throughout the Bangladeshi H9N2 genome. It is also interesting to note that the Bangladeshi H9N2 viruses contain three internal genes  from the HPAI H7N3 virus. This poses an interesting conundrum––despite carrying the molecular markers and genes from the HPAI H7N3, the Bangladeshi H9N2 viruses showed extremely limited mammalian transmission and have caused only one human infection. The viruses' endemic nature and low-virulence profile may have resulted in humans developing widespread immunity to H9N2 due to subclinical infections.62A number of studies have addressed the molecular basis of host-range specificity or adaptation to new host, but most have focused on individual gene segments such as the HA surface glycoprotein. Pathogenesis, virulence and tropism, however, are multigenic traits.25 and then test those variants in animal models. Using this approach, we can look for features of the continually evolving H9N2 viruses to see if they have reached the optimum gene-constellation level for mammalian infection and transmission and be prepared to intervene if required. However, this approach is currently controversial because it is considered a gain-of-function study. Therefore, the only current recourse is to continuously monitor the H9N2 viruses' evolution, as we have done in this study, and expand that effort.The molecular basis for host range and pathogenesis is poorly understood in H9N2 avian influenza viruses. Rather than using the naturally isolated viruses to gain insight in these areas, we need to use a reverse-genetics approach to generate H9N2 viruses with various combinations of naturally occurring mutations that have been identified in the H9N2 viruses circulating in terrestrial poultry in BangladeshMonitoring the NAI resistance of influenza viruses is a crucial part of surveillance and risk-assessment studies. NAIs complement vaccines against influenza infections; therefore, emergence of NAI resistance is a serious public health concern. Although all of the H9N2 viruses tested in this study were susceptible to NAIs due to constant reassortment with H5N1, H7N9 and H10N8, the emergence of NAI resistance remains a potential risk. Hence, the continuous monitoring of NAI resistance among H9N2 viruses is essential63 stand-alone infections in humans, swine and dogs and are also the donor of the internal genes for H7N9 and H10N8 avian influenza viruses infecting humans in China.64 As a result of the H9N2 vaccination program in China, the G1-clade H9N2 viruses have adapted and evolved tremendously, thereby posing a health concern not only for the poultry industry but also for human health. Because of this, a lot of influenza studies (past and present) involve the Chinese H9N2 viruses and not the Bangladeshi H9N2 viruses. The poultry industry in Bangladesh is very similar to that in China, with live-bird markets in which the human/animal interface is very close. However, China has an H9N2-vaccination strategy, and Bangladesh does not. Our previous study showed that the Bangladeshi H9N2 viruses have a G1-clade origin, but they are very distinct from other G1 viruses .23 Our current study shows that the Bangladeshi H9N2 viruses harbor H7N3 internal genes and multiple molecular markers throughout their genome. They also possess the potential to induce subclinical infections in ferrets but do not pose a threat to human health currently because they lack transmission potential. This finding is supported by the fact that no other mammalian H9N2 infection has been identified in Bangladesh, with the exception of a single human infection in 2011. This is extremely important, considering how prevalent H9N2 is in Bangladesh and how close the animal/human interface is. Our results suggest that the H9N2 gene constellation is currently not optimized. This emphasizes the need for further studies that monitor the viruses' evolution and aim to increase our understanding of the transmission and potential risk that these H9N2 viruses pose to humans.The G1-lineage H9N2 avian influenza viruses are of particular concern because they have been implicated in reassortment events with HPAI H5N1 viruses that resulted in human infection;


Although hearing loss may be caused by various factors, it is also a natural phenomenon associated with the aging process. This study was designed to assess the contributions of diabetes mellitus (DM) and hypertension, both chronic diseases associated with aging, as well as aging itself, to hearing loss in health screening examinees.This study included 37,773 individuals who underwent health screening examinations from 2009 to 2012. The relationships between hearing threshold and subject age, hearing threshold at each frequency based on age group, the degree of hearing loss and the presence or absence of hypertension and DM were evaluated.The prevalence of hearing loss increased with age, being 1.6%, 1.8%, 4.6%, 14.0%, 30.8%, and 49.2% in subjects in their twenties, thirties, forties, fifties, sixties, and seventies, respectively (p<0.05). Hearing value per frequency showed aging-based changes, in the order of 6000, 4000, 2000, 1000 and 500 Hz, indicating greater hearing losses at high frequencies. The degree of hearing loss ranged from mild to severe. Aging and DM were correlated with the prevalence of hearing loss (p<0.05). There was no statistically significant association between hearing loss and hypertension after adjusting for age and DM.The prevalence of hearing loss increases with age and the presence of DM. Hearing loss was greatest at high frequencies. In all age groups, mild hearing loss was the most common form of hearing loss. Aging refers to the accumulation of changes that occur from the moment of fertilization to the death of an organism, and is a universal and nonreciprocal process over time. Although the speed of aging differs in different individuals and in different organs of an individual, the aging process is inevitable.Hearing organs experience aging-associated degenerative changes, with aging being the most frequent cause of sensorineural hearing loss in adults. Hearing loss is regarded not only as a communication disorder, but as a major disease that severely impairs patient quality of life, due to social withdrawal, psychological alienation, loss of confidence, and increased depression and anxiety Degenerative diseases, including coronary artery disease, DM, hypertension, cancer, senile dementia, arthritis, and osteoporosis, interact with the aging process. Thus, good lifestyle habits can delay the appearance of chronic diseases, resulting in a better quality of life with shorter periods of sickness even with increasing life expectancy 2) individuals, using findings on hearing tests performed at a health promotion center.Although many studies have attempted to assess the correlations of DM and hypertension with hearing loss, these correlations remain unclear. Body mass index (BMI), however, has been reported to correlate with hearing loss, but there have been difficulties determining whether hearing loss correlates with BMI-associated DM and hypertension since increased BMI accompanies DM and hypertension in many patients. To better understand these relationships, this study assessed the prevalence of age-related hearing loss and whether hearing loss was associated with DM and hypertension, in a group of normal and overweight .Hearing thresholds were examined at frequencies of 500, 1,000, 2,000, 3,000, 4,000 and 6,000 Hz using an Auricle Plus audiometer  in a double-walled sound booth, in accordance with the guidelines for pure tone averages (PTA) of the Korea Occupational Safety and Health Agency (KOSHA).Frequencies were measured starting at 1,000 Hz and in the order of 2,000 Hz, 3,000 Hz, 4,000 Hz and 6,000 Hz. The subjects were re-examined at a frequency of 1,000 Hz and air conduction thresholds were measured at a frequency of 500 Hz and then at 250 Hz.Degrees of hearing were based on the standards for noise-induced hearing loss and communication disorders. Six-dimensional methods of PTAs were determined by measuring the sum of the air conduction thresholds of the right and left ears at four frequencies, 500, 1,000, 2,000 and 4,000 Hz, frequencies frequently measured clinically and in the industrial sector and calculated as /6.The average of both ears was used for analysis. The PTA of both ears at each frequency was obtained for frequency analysis. An average hearing threshold ≥26 dB was defined as hearing loss and the degree of hearing loss was categorized as mild (26∼40 dB), moderate (41∼55 dB), moderately severe (56∼70 dB) and severe (71∼90 dB) based on the standard of ISO. The average hearing threshold in each age group and the hearing threshold at each frequency were also determined Blood pressure was measured using a standardized mercury manometer after subjects had rested for 20 minutes on an empty stomach.Blood pressure was measured with an automatic blood pressure monitor; if abnormal, it was again measured manually. Based on the WHO Criteria (1978), hypertension was defined as a systolic blood pressure ≥140 mmHg or a diastolic blood pressure ≥90 mmHg.Subjects were asked to avoid vigorous exercise or alcohol consumption for 24 hours, and to maintain a fasting state for at least 8 hours prior to blood sample collection. Blood samples were collected into anticoagulant-free tubes  and analyzed with an automatic hematology analyzer .DM was defined according to American Diabetes Association criteria as a normal fasting blood glucose concentration ≥126 mg/dl or an HbA1c level ≥6.5%.The prevalence and severity of hearing loss were analyzed in each group of subjects. Subjects with hearing loss were also subclassified by its severity  and the relationship between risk factors and hearing loss in each age group was determined. As the association between hearing loss and BMI independent of DM is unclear, analysis was limited to subjects with normal BMI.To assess the risk factors associated with hearing loss, subjects were divided into two groups: those with normal hearing and those with hearing loss. Between group differences in age and prevalence rates of hypertension and DM were compared using t-tests and Chi-square tests. Univariate and multivariate logistic regression analyses were performed to determine the crude and adjusted odds ratios (ORs) of age, hypertension, DM and gender as factors associated with hearing loss. All statistical analyses were performed using IBM SPSS version 20 , with a p value <0.05 defined as statistically significant.The prevalence rates of hearing loss significantly increased with age, being 1.6%, 1.8%, 4.6%, 14.0%, 30.8%, and 49.2% in subjects in their twenties, thirties, forties, fifties, sixties and seventies, respectively (p<0.05) ; Table 1Mild, moderate, moderately severe and severe hearing loss was observed in 0.0%, 0.9%, 0.6%, and 0.0%, respectively, of subjects in their twenties; in 1.3%, 0.4%, 0.1%, and 0.03%, respectively, of subjects in their thirties; in 3.3%, 0.9%, 0.3%, and 0.1%, respectively, of subjects in their forties; in 9.5%, 2.8%, 1.1%, and 0.5%, respectively, of subjects in their fifties; in 19.9%, 7.6%, 2.2%, and 1.2%, respectively, of subjects in their sixties; and in 25.0%, 18.1%, 3.8%, and 2.2%, respectively, of subjects in their seventies , showingMean age was greater in subjects with hearing loss than in the normal hearing group .The prevalence rates of hearing loss were significantly greater in subjects with than without hypertension  and in subjects with than without DM  .Univariate analysis showed that age, DM and hypertension were significantly associated with hearing loss (p<0.05). Multivariate analysis showed that DM was an independent predictor of hearing loss (p<0.05), whereas hypertension was not p>0.05; .The aging of hearing organs is due to degenerative changes, resulting in hearing loss. Its age of onset and degree of progression are determined by both genetic and environmental factors. Aging of the hearing apparatus is characterized by bilateral symmetric sensorineural hearing loss in high frequency regions, ≥2 kHz. Moreover, males are more severely affected than females In the United States, 80% of individuals with hearing loss are aged ≥65 years, with these subjects having at least a four-fold higher incidence of hearing loss than other age groups. Moreover, some degree of hearing loss is observed in almost all subjects aged >70 years In this study, normal hearing was defined as a 0–25 dB range and hearing loss as ≥26 dB. Using these criteria, the prevalence of hearing loss increased with age, ranging from 1.6% for subjects in their twenties to 49.2% for subjects in their seventies. The most frequent degree of hearing loss was mild, although all levels of hearing loss increased with age. Abnormal findings at 6 kHz were observed in all age groups; as age increased, abnormal findings were observed in the order 4 kHz, 2 kHz, 1 kHz and 500 Hz. These findings indicate that hearing loss starts at high frequency, occurring later at the frequency of normal human speech and then at low frequency.Sound is perceived through a complicated pathway, and functional changes in the composition of this pathway weaken hearing ability with age. Although aging-associated hearing loss is thought to be associated with the aging of auditory hair cells and neurons, and the degeneration of angioid streaks, the exact mechanism of degeneration remains unclear. About 15% of elderly people have motility disorders, regardless of the presence or absence of neurologic disease. Hair cell defects, tissue degeneration, and apoptosis in the cochlear lateral wall have been detected histologically in the ears of elderly subjects. These anatomical changes in auditory organs may be caused by physiological changes that affect hearing function Aging-associated hearing loss is due primarily to genetic factors, but other factors may be involved, including exposure to noise, ear infections, smoking, hormones, gender, ototoxic substances, head trauma, cardiovascular disease , plasma hyperviscosity, DM, immune function impairment, metabolic bone disease, renal failure, endocrine medical conditions, Alzheimer’s disease, bone mineral density, ontological conditions such as Meniere’s disease and otosclerosis and as yet unidentified causes Previous studies assessing the correlation between hypertension and hearing loss differ by study subjects, study methods, and analytic methods. Most studies showed a correlation between hearing loss and aging-related hypertension but one study, in the Sudanese native population, reported no correlation The incidence of DM, a chronic disease in adults, is increasing worldwide. DM has been reported to induce progressive bilateral sensorineural hearing loss with aspects similar to presbycusis, including greater hearing loss at high frequencies. DM is frequently accompanied by complications, including neuropathy, retinopathy, nephropathy, ischemic heart disease and hypertension Studies reporting no correlation between DM and hearing threshold have found that hearing threshold at low frequency was correlated with serum glucose concentration, with hearing thresholds increasing with increasing serum glucose Many studies have reported a close correlation between DM and hearing loss, with DM patients showing a higher incidence of sensorineural hearing loss. These studies include a study in which hearing was normal during early stages of DM, whereas 30% of patients with chronic DM had hearing loss We found that the prevalence of hearing loss was significantly higher in subjects with than without DM . In addition, multivariate analysis showed that DM was a significant predictor of hearing loss (OR 1.398). The potential mechanisms of hearing loss from DM include damage to the cochlea from circulatory disorders of the inner ear; retrocochlear hearing loss caused by neuritis of the auditory nerve; the occurrence of diabetic neuropathy; and mitochondrial DNA mutations This study had several limitations. First, due to its cross sectional nature, this study assessed subjects born at different times, with different nutritional states, environments, and social, economic, and cultural backgrounds. Second, limiting subjects to health screen examinees may have introduced a selection bias. Third, various factors associated with aging were not considered, including smoking, exercise, drinking, drug use, chronic diseases, noise exposure and occupation.This study showed that the prevalence of hearing loss increases with age, as well as being higher in subjects with than without DM.


Objectives. This study analyzed the long-term hearing loss after treatment of primary nasopharyngeal carcinoma to elucidate its causal factors. Methods. Ninety-two nasopharyngeal carcinoma patients were treated with radiotherapy or chemoradiotherapy. Pure tone audiometry was performed before the therapy and annually up to 9 years after completing treatment. The hearing thresholds were corrected for age-related deterioration and compared to the results without adjusting for age. Results. The mean air and bone conduction threshold with and without correction for age-related deterioration differed significantly 2–9 years after completing radiotherapy (p < 0.05). The audiometry results with age correction showed a flattened configuration compared to the results without age correction. The total radiation dose and radiation modality showed a causal relationship with a greater incidence of hearing loss after therapy (p < 0.05). There was more deterioration in the air and bone hearing thresholds with conformal radiotherapy than intensity-modulated radiotherapy (p < 0.001). A radiation dose >72 cGy resulted in more severe hearing loss than <72 cGy (p < 0.05). Conclusion. Hearing loss after completing therapy should be corrected for age-related hearing deterioration to reveal the true extent to which the loss is a therapeutic complication. Both the radiation modality used and the dose were significantly associated with hearing loss. Nasopharyngeal carcinoma (NPC) is often located in the fossa of Rosenmüller near the opening of the Eustachian tube into the nasopharynx. Of these tumors, 95% are undifferentiated squamous cell carcinoma, which is very sensitive to radiotherapy (RT) . ChemoraWhile RT or chemoradiotherapy is the standard approach for NPC, the radiation and cisplatin ototoxicity inevitably damage the auditory apparatus –8. This Any study of HL may be erroneous if the influence of patient age on hearing status is not considered because age can affect HL . We reviNinety-two newly diagnosed cases of NPC were treated in a single medical center between June 1994 and July 2010. Audiograms were obtained before RT and at least 12 months of follow-up after completing RT. We enrolled NPC patients who were older than 18 years and had no significant preexisting HL; that is, the average bone conduction hearing threshold levels (0.5–4 kHz) were below 70 dB before RT. Patients who received additional RT for metastatic or recurrent disease, who were exposed to noise, or who received ototoxic medications other than cisplatin were excluded from the study. This study protocol was approved by the institutional review board of Chang Gung Memorial Hospital.2.Patients with stage I/IIa were treated with RT alone, while the locally advanced stage IIb-IV patients were treated with chemoradiation therapy. With updates at our institution, the radiation technique has changed from two-dimensional conformal RT (2D CRT) to three-dimensional conformal RT (3D CRT) to intensity-modulated RT (IMRT). The total radiation dose was 59.4–79.2 Gy with a mean dose of 70.86 Gy. Patients with locally advanced disease were given concurrent intravenous cisplatin chemotherapy at a mean dose of 365.57 (range 120–670) mg/mAudiometry was performed before and after RT at 1-year intervals for up to 9 years. European Committee for Standardization (CEN) data were used to obtain corrected thresholds by subtracting the loss corresponding to age and sex from the auditory threshold obtained at each frequency . For exat-test was used to determine the correlation between the possible predisposing factors and average hearing threshold deterioration. Univariate and multivariate analyses were performed with a Cox stepwise logistic regression model to identify independent prognostic indicators. A paired t-test was used to assess the serial changes in hearing with or without age adjustment. A p value less than 0.05 was considered to be statistically significant. All statistical tests were two-sided.The data were analyzed using SPSS ver. 19.0 . An independent This study included 182 ears from 92 patients. The patient characteristics and therapeutic modalities are summarized in The average hearing thresholds (not considering age-related HL or presbycusis) are shown in p < 0.05), especially at 4–8 kHz (p < 0.05). The levels of hearing deterioration were less than those without adjusting (p < 0.05). The deterioration at all frequencies tended to decrease after adjusting for age, especially at 4 kHz , while the differences were not significant at 1 year after completing RT (p = 0.16) . We found that adjusting for age decreased the deterioration of HL after completing treatment.The air conduction hearing deterioration at 0.5–4 kHz with and without age-related correction differed significantly at the 2- to 9-year follow-ups ( = 0.16) . The patp < 0.001; 14.01 dB versus 6.85 dB p < 0.001), radiation dose , and OME  were important determinants of hearing deterioration via both air and bone conduction. Radiation modality (p < 0.001) and radiation dose  remained significant in multivariate analyses. We found that deterioration in both the air and bone conduction thresholds was less in the IMRT group than in the 2D/3D CRT group 5 years after completing RT. The deterioration in hearing threshold was also greater when the total radiation dose to the primary tumor was >72 Gy.The mean hearing deterioration after adjusting for age at 0.5–4 kHz in the first 5 years was analyzed in terms of gender, age, radiation modality (2D-3D CRT and IMRT), concurrent chemotherapy, radiation dose (<72 Gy and >72 Gy), and post-RT otitis media with effusion (OME) . The radp < 0.001; 16.19 dB versus 5.91 dB p < 0.001), radiation dose , and OME  were important determinants of hearing deterioration via both air and bone conduction in the chemoradiotherapy group. Radiation modality (p < 0.001 for both air and bone) was significant in multivariate analyses. Only radiation dose (12.07 dB versus 22.67 dB p = 0.036 for air conduction) remained significant in the RT group.In order to evaluate the effect of cisplatin, we further analyzed the hearing deterioration in both RT (89 ears without cisplatin) and chemoradiotherapy (93 ears with cisplatin) groups. The radiation modality  5 years after completing RT  and chemoradiotherapy (93 ears with cisplatin) groups. The radiation modality (CRT and IMRT), radiation dose, and OME were significant via both air and bone conduction in the chemoradiotherapy group. Radiation modality remained significant in multivariate analyses. Only radiation dose was significant in the RT group. Cisplatin appeared to increase the hearing deterioration in NPC patients using a CRT technique.Chemotherapy using cisplatin did not predict HL, in agreement with previous reports  24, 26], 2624, 22 , 28. In Post-RT OME was not an independent predictor of hearing threshold deterioration. The characteristics of post-RT OME differed with the radiation dose and modality. The post-RT OME group treated with IMRT and a radiation dose <72 Gy might have had less severe hearing threshold deterioration. With IMRT, the dose to the Eustachian tube was <52 Gy, reducing the incidence of post-RT OME and severity of air and bone conduction threshold (0.5–4 kHz) deterioration , 30. YouSome studies have observed a persistent association between the presence of post-RT OME and HL , 32. The per se on hearing status were not considered; again, these factors affect HL.We found no significant correlation between age and gender and post-RT HL data adjusted or not for age-related threshold deterioration. The effects of age and gender on the development of RT-induced HL were inconsistent in previous reports . Some auThis study was retrospective in nature, and several potential limitations should be mentioned. First, follow-up was performed after various intervals by different physicians, and it is likely that the follow-up protocols differed greatly. For example, the instruments used for measurement will have varied . In addition, the RT techniques will have varied. NPC patients may have died or become lost to follow-up. Second, data incompleteness and differences in the way data were annotated in databases and medical charts render it difficult to define the cause of HL after RT. Moreover, all the evaluation of long-term HL after treatment of primary NPC was performed at a single center. We believe that these factors have affected all cases similarly, merely rendering our estimations imprecise; we do not believe that we have exaggerated or minimized such estimations. Patients with possible confounding factors, such as significant preexisting HL, use of ototoxic medications other than cisplatin, or exposure to noise, were excluded from the study.There is a concern that the issue of cisplatin, an ototoxic drug, could confound the data collected. Since late stage NPC patients treated with cisplatin were included in this study, it was difficult to separate an aging and radiation in patients with cisplatin versus no cisplatin. Cisplatin interferes with the overall findings and conclusions reached.After adjusting for age-related hearing threshold deterioration, HL owing to the treatment of NPC was present clinically at least 12 months after RT and involved all frequencies. Therefore, it is essential to adjust for patient age when determining the true change in hearing following RT or chemoradiotherapy. IMRT and total radiation dose less than 72 Gy tended to result in less deterioration in hearing.The hearing thresholds of air conduction at 0.25, 0.5, 1, 2, 4, and 8 kHz were measured and adjusted (Supplementary Table 1). The air (0.25–8 kHz) and bone (0.5–4 kHz) conduction hearing deterioration with and without age-related correction at the 1- to 9-year follow-up after RT were listed in Supplementary Table 2 and 3, respectively. The mean air and bone conduction hearing deterioration at 0.5–4 kHz with and without age-related correction were measured (Supplementary Table 4).


Introduction: The aim of this study was to measure the haemodynamic responses to a etomidate-propofol combination used for anaesthesia induction and to compare the haemodynamic responses with the separate use of each drug.Methods: The patients were randomly divided into three groups as group P , group E  and group PE . For each patient, the times of measurement of the heart rate (HR) and mean arterial pressure values were defined as baseline, after the induction, before the intubation, immediately after the intubation and 1, 2, 3, 4, 5 and 10 minutes after the intubation.Results: In all 3 groups, a significant decrease in MAP values were seen at T2 and T3 compared to the baseline values, and this decrease was greater in group P compared to that in group E and PE . A significant increase was seen in all 3 groups in the mean arterial pressure (MAP) value at T4 after the intubation. When the groups were compared with each other, this increase was greater in group E than in the other two groups .Conclusion: Etomidate-propofol combination may be a valuable alternative when extremes of hypotensive and hypertensive responses due to propofol and etomidate are best to be avoided. However, when intravenous induction drugs are used as a single hypnotic agent, haemodynamic side-effects are frequently observed.4 Etomidate is a hypnotic agent with minimal effects on the cardiovascular system. It does not cause histamine expression and has no analgesic properties. Etomidate’s side-effects are primarily injection pain, myoclonus, superficial thrombophlebitis and a high incidence of nausea and vomiting.3 Previous studies have also reported that etomidate did not prevent the sympathetic response to laryngoscopy and intubation at a sufficient level.5Propofol is a frequently-used intravenous anaesthetic with an effect of rapid onset and short duration. During anaesthesia induction with propofol, often seen side effects are injection pain and a fall in arterial blood pressure.The hypothesis of this study was that with the use of average doses of etomidate and propofol together, the haemodynamic deterioration after the anaesthesia induction and endotracheal intubation would decrease. With the aim of testing this hypothesis, consecutive doses were used within the permitted limits in the clinical use of propofol and etomidate in anaesthesia induction, and the effects on the haemodynamic response to intubation were measured. Primary objective was the comparison of the haemodynamic changes created with the etomidate-propofol combination and the sole use of each drug. Secondary aims were defined as the incidence of injection pain and myoclonus.https://clinicaltrials.gov/; NCT02186990). The study comprised a total of 90 patients, aged 18-65 years, who were American Society of Anesthesiologist (ASA) I-II risk group who were to undergo elective surgery with endotracheal intubation under general anaesthesia in Ordu University Research and Training Hospital between in May-August 2014. A written informed consent was obtained from all patients. Patients were excluded if they had any allergy to the medications to be used in the study, chronic use of analgesia or sedatives, body mass index (BMI) > 25 kg/m2, anticipated difficult intubation , hypertension or cardiovascular disease.Approval for the study was granted by the Local Ethics Committee (2014/06) and it was recorded in the Clinical Trials (2 saturation (SpO2) and end-tidal CO2 monitoring  were applied. Neuromuscular monitoring was applied with a TOF Watch SX  device with electrodes placed on the ulnar nerve line and evaluation was made from contractions of the adductor pollicis muscle.Patients were not administered with any premedication drugs and on admission to the operating room, electrocardiography (ECG), non-invasive blood pressure, peripheral O-1 via a 20 G venous cannula in the back of the non-dominant hand. Using a computer generated sequence of numbers and a sealed envelope technique, patients were randomly divided into 3 groups: Group P (n = 30) was administered propofol, group E (n = 30) was administered etomidate and group PE (n = 30) was administered etomidate-propofol (etofol) combination for anaesthesia induction.Lactated Ringer’s solution infusion was started at 10 mL kg-1 fentanyl to all patients in anaesthesia induction, patients in group P were administered 2.5 mg kg-1 propofol , group E, 0.3 mg kg-1 etomidate , and group PE, 1.25 mg kg-1 propofol and 0.15 mg kg-1 etomidate in separate injectors. The medications were given at the rate of 1 mL s-1 and in group PE, the venous cannula was washed out with saline for 5 seconds between medications. When loss of consciousness was obtained (eyelash reflex loss), 0.6 mg kg-1 rocuronium was administered and when no response was obtained to the train-of-four (TOF) stimulus with the TOF-guard device, the patient was intubated by the orotracheal route. Following the intubation, the patients were ventilated to preserve end-tidal CO2 pressure between 35-40 mm Hg and anaesthesia was maintained with 2% sevoflurane in a 50% O2/air mixture.After administration of 1 mcg kg6 Values of more than 15 000 mm Hg min-1 are accepted as an increased cardiac risk.7The measurement times for the parameters of heart rate (HR) and mean arterial pressure (MAP) were T1, basal values; T2, after induction; T3, before intubation; T4, immediately after intubation; and T5, T6, T7, T8, T9 and T10 at 1, 2, 3, 4, 5, and 10 minutes after intubation. All the measurement intervals, ‘rate pressure product’ (RPP) values were calculated. RRP is defined as the product of systolic blood pressure and HR values.8 Myoclonus was evaluated in all patients by the same researcher (NT) using the presence of muscular activity: 0 = no myoclonus, 1 = myoclonus present.In all patients, injection pain was evaluated by the same researcher (NT) by measurement on a 4-point scale as described in previous studies: 0 = no pain, 1 = verbal complaint of pain, 2 = withdrawal of the arm, 3 = both verbal complaint and withdrawal of the arm.9 when MAP at the first minute of intubation is taken as the main result, in the event of at least 30 patients in each group, it was calculated that in respect of haemodynamic parameters, a 10% difference could be determined between the groups at 80% power and 5% significance  .According to the evaluation made on the basis of a previous study,P  ≤  0.05 was accepted as statistically significant.Data obtained in the study were analysed with SPSS 16.0 . Descriptive statistics were stated as mean ± standard deviation for continuous variables and as number and percentage for nominal variables. Distribution analysis was made with the Kolmogorov-Smirnov test. Age, body mass index (BMI), HR, MAP and RPP were evaluated with one-way analysis of variance (ANOVA) and post-hoc tests with Bonferroni correction. The chi-square test was used for categorical data such as gender, ASA physical status, injection pain and myoclonus. A value of P > 0.05; The data of 90 patients were evaluated in the study . No statP < 0.001) and group PE (P < 0.01).The changes in the MAP values according to the times are shown in P < 0.001) and group PE, (P < 0.01).At the T4, MAP values of all groups were significantly increased compared to the basal values. In the comparison between the groups at T4, the MAP values of the group E were statistically significantly higher than those of group P  and of group PE at T7 and T9 (P < 0.05 and P < 0.05).In group P, MAP values were found to be significantly lower than those of group E at T5, T6 and T7 .The changes in the HR values according to the measurement times are shown in P < 0.01 for group E, P < 0.05 for group PE; at T3, P < 0.01 for group E and PE). At T4, T5 and T6, the RPP values of the group E were found to be significantly higher compared to those of group P and PE (P < 0.01). The number of patients with an RPP value over 15000 mm Hg min-1 was 1 (3%) in the group P, 7 (23%) in group E and 2 (7%) in group PE. The difference between the propofol and etomidate groups was statistically significant (P < 0.05).The RPP values according to the measurement times are shown in P < 0.05; No statistically significant difference was determined between the groups in respect of injection pain. A significant difference was determined between group P and group E in terms of myoclonus incidence  using propofol, etomidate and a etomidate-propofol combination, after the administration of 2 mcg kg-1 iv fentanyl, one group was given 2.5 mg kg-1 propofol, one group, 0.3 mg kg-1 etomidate, and one group, 1 mg kg-1 propofol + 0.1 mg kg-1 etomidate. LMA placement was made after the loss of eyelash reflex and no response to verbal commands. The main finding of the study was that more stable haemodynamics were provided by etofol compared to propofol and etomidate. Although the doses of both drugs are reduced in the etomidate-propofol combination, it was reported that a more stable haemodynamic state and better conditions for LMA placement were provided.14Etomidate is one of the iv anaesthetics used in anaesthesia induction, either alone or in combination with other anaesthetic drugs.15 haemodynamics, myoclonus and injection pain were evaluated with propofol and etomidate at doses of the same bispectral index value as etomidate-propofol combination in the same injector in anaesthesia induction. The etomidate-propofol combination in that study was provided at a 1:1 ratio of 1% propofol (20 mg mL-1) and etomidate (2 mg mL-1). In the anesthesia induction, the medications were applied by titration to provide at a target BIS value of 40. From the results of the study, it was reported that anaesthesia induction with a combination of etomidate and propofol provided a pain-free injection, lower rate of myoclonus, and in comparison with propofol and etomidate used alone, achieved a quicker induction and better haemodynamic stability.In a study by Saricaoglu et al,17 In a study of 4096 patients in which hypotension following general anaesthesia induction was evaluated, risk factors for hypotension were defined as ASA III-IV risk group, age of ≥50 years, baseline MAP of <70 mm Hg, the use of propofol for anaesthesia induction and the use of a high dose of fentanyl in induction. To avoid severe hypotension in patients aged over 50 years with ASA III risk scores or more, an alternative to propofol should be considered in anaesthesia induction and it was particularly recommended that propofol is avoided in cases with baseline MAP of <70 mm Hg.18In cardiac and non-cardiac surgery, there is evidence related to undesired results during general anaesthesia independent of hypo or hypertension in the patient.19In a study comparing propofol+ketamine and propofol+etomidate combinations in elderly patients, it was found that as the application of propofol prevented haemodynamic changes, both ketamine and etomidate were equally effective.3 Etomidate reversibly inhibits 11-β-hydroxylase and prevents the return of 11-deoxycortisol to cortisol. Single-dose etomidate inhibits 11-β-hydroxylase in 5-8 hours postoperatively.20 Another significant disadvantage of etomidate is that it has no analgesic effect, it cannot effectively reduce the temporary sympathetic response to endotracheal intubation.22 This situation may cause a short but uncomfortable attack of hypertension and tachycardia. Even though this hyperdynamic response is short, it causes an increase in intracranial pressure and increased myocardial workload.23 In addition to these known side-effects of etomidate, it has been reported that occasionally ventricular tachycardia and fibrillation may be seen.24Besides providing good cardiovascular stability, etomidate may also cause nausea and vomiting, injection pain, myoclonus and side-effects on the endocrine system.25 which used propofol and etomidate in general anaesthesia induction accompanied by BIS monitoring, the MAP, cardiac index (CI) and systemic vascular resistance index (SVRI) values of 48 patients were compared. The haemodynamic data were found to be higher in the etomidate group up to 7 minutes after intubation. A significantly high level of hypotension incidence was found in the propofol group and a significantly high level of hypertension incidence in the etomidate group. Compared with etomidate, the use of propofol was determined to have caused less hypertension and tachycardia after intubation. In the current study, the MAP values after induction in the propofol group were significantly lower than those of the other two groups. Following intubation, the MAP and HR values of the etomidate group were statistically significantly higher than those of the other two groups. These results confirm with those in literature.In a study by Möller et al26In another study comparing the use of propofol and etomidate in anaesthesia induction, it was reported that when opioid and/or benzodiazepine support is not suitable because of a negative cardiovascular profile, myoclonus and poor mask ventilation, etomidate may be suitable in induction.-1), the ideal fentanyl dose was 5-10 mcg kg-1 to prevent a haemodynamic response to laryngoscopy and intubation.27 However, it can be predicted that the use of such a high dose of fentanyl may cause increased hypotension and nausea and vomiting.Another study reported that after anaesthesia induction with etomidate (0.3 mg kg28 a comparison was made of propofol (2 mg kg-1), thiopental (5 mg kg-1) and etomidate (0.3 mg kg-1) in anaesthesia induction. A statistically significant increase was determined in systolic and diastolic arterial pressure and HR in the etomidate and thiopental groups after intubation and the highest rates of complications were reported in the etomidate group.In a study by Muriel et al,30In two studies which compared propofol, thiopental and etomidate induction in intubation without muscle relaxant, as appropriate conditions could not be provided in the etomidate group, the study was prematurely terminated.31In another study, the haemodynamic response to orotracheal intubation was evaluated following anaesthesia induction with midazolam and etomidate. Although the systolic and diastolic pressures and RPP values were found to be lower in the midazolam group, it was reported that neither of the induction agents could prevent the haemodynamic response to intubation.6 In the current study, the RPP values of the etomidate group after intubation were statistically significantly higher than those of the other two groups. This was evaluated as an indicator that the sympathetic response formed after laryngoscopy and intubation could not be adequately prevented with etomidate.It is known that RPP is an indicator of myocardial stress and maximal oxygen consumption and is the best indirect measurement method of myocardial oxygen consumption.32 In the current study, myoclonus was determined at 20% in group E and 9% in group PE. These low rates of myoclonus are thought to be due to premedication with fentanyl. Previous studies have reported the incidence of myoclonus with fentanyl use at 8% to 40%.33In patients not receiving premedication in anaesthesia induction with etomidate, myoclonus incidence has been reported at 50%-80%.34 With fentanyl premedication, rates such as 40%, 19% and 8% have been reported.37 Reported rates of 50%-60% of etomidate injection pain are also reduced with fentanyl premedication.21Injection pain is a significant clinical problem in both propofol and etomidate use. In the current study, the incidence of injection pain was 27% in group P, 13% in group E and 10% in group PE, with no statistically significant difference between these rates. In literature, propofol injection pain has been reported at rates of 40%-86%.15 comparing propofol, etomidate and etofol in anaesthesia induction, injection pain in the etofol group was found to be lower than in the other two groups and myoclonus incidence was lower than in the etomidate group. The incidence of injection pain was reported as 83.8% in the propofol group and as 63.2% in the etomidate group. Myoclonus incidence was determined as 93.4% in the etomidate group. The reason for the high rates of these results compared to the results of the current study is thought to be that no premedication was administered in the Saricaoglu study.In a study by Saricaoglu et al15 Due to the risk of propofol contamination in particular, it has been reported that it is necessary to apply strict aseptic techniques during preparation and application.38 Severe infection tables have been reported because of propofol contamination.39 In the current study, separate injectors were used because of the increased risk of contamination while preparing the combination.In literature, propofol and etomidate mixed in the same injector have been used.40 In a study by Saricaoglu et al15 propofol, etomidate and etofol were administered in a manner that the BIS value would be 40. The doses used in the current study were similar to those used in the Saricaoglu study.Limitations of the current study were primarily that BIS measurement was not applied to evaluate loss of consciousness and to determine the depth of anaesthesia. It has been reported that for etomidate, a BIS value of 50 is sufficient for conscious movement and absence of memory in tracheal intubation.20Another limitation of the current study is that plasma cortisol and adrenocorticotropic hormone level were not measured. However, as the use of single-dose etomidate causes adrenocortical suppression, it has been reported to be temporary and not of clinical importance.In the literature there are 3 studies on the etomidate-propofol combination. The first of these is the study by Sarıcaoglu et al. comparing propofol, etomidate and etofol in terms of effects on hemodynamic changes only during anesthesia induction. Another is a study comparing effects of etomidate and ketamine added to propofol by Hosseinzadeh et al. The final study is again by Hosseinzadeh et al and compares propofol, etomidate and etofol in terms of hemodynamic changes during LMA insertion. Our study compared etofol with etomidate and propofol, not only during anesthesia induction but after tracheal intubation. In conclusion, with the etomidate-propofol combination, a milder haemodynamic response was determined compared to propofol and etomidate used alone, both at anaesthesia induction and after the intubation. Etofol can be a valuable alternative in patients where cardiovascular fluctuations are not wanted.Ethics committee approval was received for this study from Ethics Committee of Harran University (2014/06). Also, written informed consent was obtained from patients who participated in this study.Authors declare no conflict of interest in this study.


GJB2) mutations are responsible for ~50% of non-syndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate that congenital deafness mainly results from cochlear developmental disorders rather than hair cell degeneration and endocochlear potential reduction, while late-onset hearing loss results from reduction of active cochlear amplification, even though cochlear hair cells have no connexin expression. However, there is no apparent, demonstrable relationship between specific changes in connexin (channel) functions and the phenotypes of mutation-induced hearing loss. Moreover, new experiments further demonstrate that the hypothesized K+-recycling disruption is not a principal deafness mechanism for connexin deficiency induced hearing loss. Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), and Cx43 (GJA1) mutations can also cause hearing loss with distinct pathological changes in the cochlea. These new studies provide invaluable information about deafness mechanisms underlying connexin mutation-induced hearing loss and also provide important information for developing new protective and therapeutic strategies for this common deafness. However, the detailed cellular mechanisms underlying these pathological changes remain unclear. Also, little is known about specific mutation-induced pathological changes in vivo and little information is available for humans. Such further studies are urgently required.Hearing loss due to mutations in the connexin gene family, which encodes gap junctional proteins, is a common form of hereditary deafness. In particular, connexin 26 (Cx26,  Gap junctions are intercellular channels that connect the cytoplasm of adjacent cells, providing a direct intracellular conduit for intercellular communication. A gap junctional channel is formed by two hemichannels; each hemichannel is composed of six subunits , gap junction channels possess a relatively large pore size (~10–15 Å) and can allow passage of ions, cell signaling molecules, and small molecules up to ~1.5 kDa  mutations are a common genetic cause for non-syndromic hearing loss and are responsible for ~50% of non-syndromic hearing loss in children  Most mutants cannot correctly track to the cell surface to form functional gap junction channels , the spiral ligament, the spiral limbus, and other cochlear non-sensory cells and structures , GJB6 (Cx30), GJB3 (Cx31), GJC3 (Cx29), and GJA1 (Cx43), have been identified in the cochlea  was initially reported on bone of the otic capsule and on cells lining the inside of the bony wall in adult animals  are anions. This may be a reason why Cx26 but not Cx30 mutations induce high-incidence of hearing loss and why Cx30 knockin cannot restore hearing of Cx26 deficient mice  cochlear developmental disorders, (ii) hair cell and SG neuron degeneration, (iii) EP reduction, and (iv) impairment in active cochlear amplification . By P10, the EP and Ks Ehret, . Hearings Ehret,   between the intracellular space and the intrastrial space, i.e., +115–125 mV in the intrastrial space with respect to normal extracellular space. Finally, this positive intrastrial potential eventually leads to positive EP (+100–120 mV) in the endolymph in the scala media  in the cochlear endolymph in the scala media  threshold was increased by ~35 dB sound pressure level (SPL) in these mice, and EP was reduced by 20–40% . Finally, similar to the K+-recycling disruption hypothesis, our recent finding that after deletion of Cx26 in the cochlea mice can still retain normal hearing at young ages , multiple connexin genes GJB6 (Cx30), GJB3 (Cx31), GJC3 (Cx29), and GJA1 (Cx43), have been identified in the cochlea. Mutations of these connexin genes can also cause hearing loss, even if they are rare.In addition to GJB6 (Cx30) gene is associated with non-syndromic hearing loss through either homozygous deletion of Cx30 or digenic inheritance of a Cx30 deletion and a Cx26 mutation  mutations were also initially linked to non-syndromic autosomal recessive deafness  in Cx26 deficient mice can restore hearing (Degen et al., It has been reported that restoration of Cx26 expression by knockin of an external Cx26 copy in the Cx30 KO mouse line can rescue its hearing, because the Cx26 expression level in the Cx30 KO mice was also dramatically reduced (Ahmad et al., Recently, it has also been reported that a genetic approach to restore Cx26 expression in the cochlea in Cx26 KO mice after birth can preserve both hair cells and SG neurons but cannot restore hearing function (Yu et al., +-recycling disruption is not a principal deafness mechanism for Cx26 deficiency induced hearing loss (Chen et al., Cx26 mutations are the major cause of hereditary deafness and can induce congenital deafness and late-onset hearing loss (Castillo and Castillo, However, the cellular mechanisms underlying Cx26 deficiency induced cochlear developmental disorders remain unclear. The mechanisms whereby Cx26 deficiency in the supporting cells influences OHC electromotility and active cochlear amplification are also unclear. Moreover, it is unknown whether specific mutations of connexins will produce the same pathological changes as connexin knockout mice. Finally, little is known about pathological changes in the human cochlea, and it is unclear whether the observed pathological changes in mice are similar to humans.in vivo. Recently, it has been reported that administration of vitamins A, C, and E and magnesium ameliorated progressive hearing loss in a child with Cx26 mutation (Thatcher et al., Creation of specific connexin mutation knockin mouse models and investigation of pathological changes in the human cochlea are urgently required. The knockin transgenic mice can also provide important information about the relationship between mutated protein expression and absence of protein expression The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Occupational hearing loss is an increasingly prevalent occupational condition worldwide, and has been reported to occur in a wide range of workplaces; however, its prevalence among workers from municipal solid waste landfills (MSWLs) remains less clear. This study aimed to investigate the occupational hearing loss among Chinese MSWL workers.A cross-sectional study of 247 workers from 4 Chinese MSWLs was conducted. Noise and total volatile organic compounds (TVOCs) levels at worksites were determined. We conducted hearing examinations to determine hearing thresholds. A worker was identified as having hearing loss if the mean threshold at 2000, 3000 and 4000 Hz in either ear was equal to or greater than 25 dB. Prevalence of occupational hearing loss was then evaluated. Using unconditional Logistic regression models, we estimated the odds ratios (ORs) of MSWL work associated with hearing loss.According to the job title for each worker, the study subjects were divided into 3 groups, including group 1 of 63 workers without MSWL occupational hazards exposure (control group), group 2 of 84 workers with a few or short-period MSWL occupational hazards exposure, and group 3 of 100 workers with continuous MSWL occupational hazards exposure. Both noise and TVOCs levels were significantly higher at worksites for group 3. Significantly poorer hearing thresholds at frequencies of 2000, 3000 and 4000 Hz were found in group 3, compared with that in group 1 and group 2. The overall prevalence rate of hearing loss was 23. 5%, with the highest in group 3 (36.0%). The OR of MSWL work associated with hearing loss was 3.39 .The results of this study suggest significantly higher prevalence of hearing loss among MSWL workers. Further studies are needed to explore possible exposure-response relationship between MSWL occupational hazards exposure and hearing loss. Occupational hearing loss is an increasingly prevalent occupational condition and occurs across many countries. It can be induced by several factors in workplace, such as noise, organic solvents, and other ototoxic substances. . Noise iOccupational hearing loss has been reported to occur in a wide range of workplaces, including printing, painting, manufacture of drug, foods and metal mining –8. HowevAs the main risk factor of hearing loss, noise in MSWLs is mainly generated by the large machineries and can be even over 90 dB that much higher than OSHA noise standard 1910.95 (85 dB) . HoweverHere, we carried out a cross-sectional study to conduct environmental monitoring at MSWL work sites, administrated audiometric testing and collected data through questionnaires of MSWL workers, aiming to investigate the occupational hazards, including noise and TVOCs, at MSWLs and evaluate the occupational hearing loss among MSWL workers.In this cross-sectional study, we investigated 4 Chinese MSWLs that were located in Shenzhen (Guangdong province), Shanghai, Harbin (Heilongjiang province) and Hangzhou (Zhejiang province) city, respectively. A total of 298 workers who had worked for at least 1 year were involved in our investigation. All these workers underwent and passed health examinations (including hearing examination) before their work at these landfills. We divided the subjects into 3 groups based on the specific work sites in the MSWLs where they mainly worked: group 1, general workers without occupational hazards (control group), including administration staff, engineering designers, guards, barbers, cooks, financial staff, cleaners, non-garbage truck drivers, etc.; group 2, workers with only a few or short-period occupational hazards associated with landfilling during their work shift, including environmental monitoring workers, laboratory assistants, road-menders, field directors, repairmen, technicians, safety managers, etc.; group 3, workers with continuous occupational hazards associated with landfilling, including bulldozer drivers, garbage truck drivers, excavating-machine operators, compacting machine operators, etc.A specified questionnaire was implemented by trained investigators to collect data on demographic information , smoking status, drinking habit, etc.  Smoking Environmental noise levels of 4 MSWLs determined by work site environmental monitoring using portable sound level meter . The time-weighted noise levels for drivers or operators of large machineries were measured by attaching the Quest EDGE-4 Personal Noise Dosimeter during their work.Before hearing examination, workers with local ear problems  were excluded via local otoscopic examinations by a doctor. Workers eligible for hearing examination were asked for their work history, and if they had ever been diagnosed as hearing loss or deafness due to drugs, genetic disorders or any other risk factors. An audiometer  with a middle ear analyzer  was then used to measure the pure-tone air and bone conduction hearing thresholds at frequencies of 500, 1000, 2000, 3000, 4000 and 6000 Hz. The audiometric testing was conducted in a sound-isolated room and operated by a certificated occupational health doctor. All the candidate subjects were measured after a rest of at least 10 minutes in the morning prior to their work. The measured hearing thresholds were adjusted for gender and age based on the China National Standard “Diagnosis of occupational noise-induced deafness” (GBZ 49–2007). Under the current OSHA Standard (29 CFR 1904.10), a worker was defined as hearing loss if the audiogram identified a hearing threshold value of equal or greater than 25dB for the mean combined value of the 2000, 3000, and 4000 Hz in one or both ears ,18.Variables of age, years of education and duration of work were dichotomized based on their distributions in studied subjects. Continuous variables, such as noise level, for different groups of subjects with normal distribution and equal variance were described using means and standard deviations, and compared using Analysis of Variance (ANOVA) followed by the least significant difference (LSD) test for pair-wise comparisons. Continuous variables that did not met the conditions for ANOVA were described by median and interquartile range (IQR), and compared using Kruskal-Wallis test followed by Nemenyi test for pair-wise comparisons . CategorP values are two-sided.Selected characteristics , noise levels, result of audiometric testing and prevalence of hearing loss were compared among group 1, group 2 and group 3 of the study subjects. Odds ratios (OR) and the corresponding 95% confidence intervals (CIs) for different group as well as potential confounders associated with hearing loss were estimated using multivariate unconditional Logistic regressions. Because there was no female worker in group 3 of subjects, we also estimated ORs and 95%CIs by restricting the regression analysis in male workers only. All analyses were conducted with SAS version 9.1. All given P < 0.05). Ratios of regularly smoking and drinking were significantly higher in group 3, compared to both group 1 and group 2 (P < 0.05). The education level of workers in group 3 was significantly lower than that in group 1 and group 2 (P < 0.05). These descriptive statistics and comparisons did not change materially for male workers only.Of the 298 landfill waste landfill workers, all of them completed the questionnaire for demographic information; 2 (0.7%) of them did not finish the questionnaire for work history; 49 (16.4%) of them were not administrated audiometric testing; 2 (0.7%) of them suffered from significant hearing loss due to ototoxic drug use. No workers claimed any genetic risk for hearing loss. The 2 workers without work history did not finish audiometric testing neither; therefore, the 298 potential study subjects were reduced to 247 with a participant rate of 82.9%. Proportion of male workers for the 298 workers and the selected 247 workers were 81.0% and 80.6%, respectively; while the median age for the two populations were 39.0 and 38.0 years, respectively. Distributions and differences among subjects of selected characteristics were shown in P < 0.05), though there was no significant difference between group 1 and group 2. Of the 56 noise samples, 3 (5.4%) of them were over 85 dB, all of which were from group 3 (8.8%). The mean time-weighted noise exposure of bulldozer drivers and compacting machine operators were 95.1 dB and 91.1 dB, respectively. As shown in P < 0.05).P > 0.05); however, the hearing thresholds of group 3 was significantly higher than that of both group 1 and group 2 (P < 0.05). Similarly, the mean combined value of hearing thresholds at 2000, 3000 and 4000 Hz for workers from group 3 was significantly higher than that from group 1 and group 2 (P < 0.05).P < 0.05) except for worker with experience of smoking and other landfill-related work.Based on the OSHA Standard, there were 58 hearing loss cases in all investigated workers, resulting in a prevalence rate of 23.5%. All these hearing loss cases were sensorineural hearing loss cases. The number of hearing loss cases in group 1, group 2 and group 3 were 10 , 12 (14.3%), and 36 (36.0%) respectively. The number and prevalence of hearing loss by selected characteristics in different groups of the subjects was showed in The multivariate unconditional Logistic regressions gave estimated odds ratios and the corresponding 95% CIs for groups and potential confounders, including different groups, gender, age, years of education, drinking, smoking status, duration of work and other landfill-related work . HearingThe results of our study suggested significantly higher prevalence of hearing loss among MSWL workers. Noise and TVOCs might be potential risk factors for MSWL workers to develop hearing impairment, although their causal relationship needs to be further investigated by conducting quantitative exposure assessment and exposure-response analyses. Nonetheless, our data provided a primary result regarding the prevalence of hearing loss among MSWL workers.The hearing loss among MSWL workers may be mostly attributable to the high noise exposure. There are several possible explanations to address this. One is the higher noise level for MSWL workers. The large machineries that were used to dig, transport and compact landfills can generate noise level higher than 85 dB (group 3). The result of environmental noise monitoring showed that the mean noise level was 70.1 dB which was significantly much higher than that in work sites of group 1 (57.5 dB) and group 2 (60.7 dB). The other reason is the continuity of noise exposure (group 3). The exposure pattern of group 3 was continuously exposed to a relatively constant level of noise during work time. Continuous noise exposure throughout the workday and over years can be more damaging than interrupted exposure to noise that permits the ear to have a rest period (group 2) . The laset al. found that combined exposure to mixed organic solvents  and noise could exacerbate hearing loss, especially high-frequency  hearing loss, in workers [et al. found elevated hearing impairment compared to workers exposed to noise only, suggesting that the solvent exposure contributed process of hearing loss [Meanwhile, other occupational hazards might have contributed to the higher prevalence of hearing loss among MSWL workers. Besides noise, our previous study showed that the main occupational hazards also included respirable dust, heat, ozone, ammonia, methane, hydrogen sulfide and TVOCs . A study workers . By evaling loss . In thisVarious studies have shown that age, smoking and some other factors are associated with higher prevalence of hearing loss ,22,23. ISeveral limitations should be addressed for our study. One is that the MSWLs in our study did not have sufficient data on previous occupational noise exposure. In the analysis, we used our monitoring data to evaluate noise level, and assumed that the noise level had not changed overtime. This might have led to inaccuracy as regards to the noise exposure; however, the noise levels for MSWL workers, especially drivers of large machineries, were not likely to change dynamically. Second, the sample size is relatively small. Unlike other studies , odds raThe present study demonstrated increased prevalence occupational hearing loss among MSWL workers. Possible reasons may include exposure to noise and TVOCs. Further studies needs to be conducted to better understand associations between occupational risk factors and hearing loss. Hearing conservation program should be considered to implement in workplaces of MSWLs. Health promotion should also be conducted to help MSWL workers protect themselves from excess noise exposure, such as regular health examinations and personal protective equipment use.