Ginkgo leaves are always resources for flavonoids pharmaceutical industry. However, the effect of the elevation and tree age changes on flavonoid biosynthesis have not been detailly explored in Ginkgo leaves. In addition, whether these environmental pressures have similar effects on the biosynthesis of other non-flavonoids polyphenolics in phenylpropanoid biosynthesis is not known at present. In this research, de novo transcriptome sequencing of Ginkgo leaves was performed coupled with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analyses to obtain a comprehensive understanding of the influence of elevation and tree age on phenylpropanoid biosynthesis. A total of 557,659,530 clean reads were assembled into 188,155 unigenes, of which 135,102 (71.80%) were successfully annotated in seven public databases. The putative DFRs, LARs, and ANRs were significantly up-regulated with the increase of elevation in young Ginkgo tree leaves. The relative concentration of flavonoid derivatives with high parent ion intensity was likely to imply that the elevation increase promoted the biosynthesis of flavonoids. Complex gene variations involved in flavonoid biosynthesis were observed with the tree age increase. However, flavonoid derivatives analysis predicted that the rise of tree age was more likely to be detrimental to the flavonoids manufacture. Otherwise, multiple genes implicated in the synthesis of hydroxycinnamates, lignin, and lignan exhibited fluctuations with the elevation increase. Significantly up-regulated CADs and down-regulated PRDs potentially led to the accumulation of p-Coumaryl alcohol, one of the lignin monomers, and might inhibit further lignification. Overall, the putative DFRs seemed to show more considerable variability toward these stress, and appeared to be the main regulatory point in the flavonoid biosynthesis. Light enhancement caused by elevation increase may be the main reason for flavonoids accumulation. Flavonoid biosynthesis exhibited a greater degree of perturbation than that of hydroxycinnamates, lignins and lignans, potentially suggesting that flavonoid biosynthesis might be more susceptible than other branch pathways involved in phenylpropanoid biosynthesis. This research effectively expanded the functional genomic library and provide new insights into phenylpropanoid biosynthesis in Ginkgo. Phenylpropanoids biosynthesis pathway functions as a natural factory to produce a variety of secondary metabolites in response to biological and abiotic stimuli . Derivedp-coumaric acid. Then, 4-coumaroyl CoA ligase (4CL) catalyzes p-coumaric acid into p-coumaroyl-CoA, which is a crucial branch point leading to the generation of flavonoids, lignins and lignans  to yield cinnamic acid . In the  lignans .Arabidopsis (Plants have evolved a variety of antioxidant systems to cope with reactive oxygen species (ROS) generated under ambient pressures . As an ibidopsis . Lignansbidopsis . Howeverbidopsis .Ginkgo biloba (Ginkgo) is widely used as traditional Chinese medicine for centuries, especially on asthma and cardiovascular disease  were chosen to collect mature leaf samples (* growth factor) proposed by the International Society of Arboriculture in the United States for estimating the age of trees, it can be inferred that the diameter at breast height (DBH) positively correlates with the age of the trees in the same (or similar) environment. So, the DBH (converted from trunk circumference) was used as a measure of the relative extent of tree ages. The elevation and the DBH were determined by Global Positioning System (GPS) instrument and tape measure respectively. All samples were divided into four groups – low elevation and young age (LY), high elevation and young age (HY), low elevation and older age (LO), high elevation and older age (HO). Each group involved three tree individuals. Each sample was mixed by thirty leaves of similarly size and health, which were cut off from the sunny side 5 m above the ground of the same tree  according to the manufacturer’s instruction coupled with genomic DNA digesting with DNase I. Then 1% agarose gel electrophoresis was used to monitor RNA degradation and contamination. RNA purity, concentration and inte- grity were measured by NanoPhotometer® spectrophotometer , Qubit 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system . Dynabeads Oligo (dT)  was used to isolate mRNA from total RNA. Then the NEB Next® Ultra RNA Library Prep Kit for Illumina®  was used to construct 12 cDNA libraries following manufacturer’s recommendations. After some necessary processes  and the terminal quality assessment on the Agilent Bioanalyzer 2100 system, the library preparations were sequenced on an Illumina Hiseq PE150 platform and paired-end reads were generated.Total RNA extraction was carried out by E.Z.N.A.1  enrichment analysis of the DEGs based on the Wallenius non-central hyper-geometric distribution (The assembled transcriptome spliced by Trinity was set as the reference sequence (ref) . The cleribution . KOBAS . Then quantitative PCR experiments were performed on Bio-Rad iQ5 Optical System  with KOD SYBR® qPCR Mix Kit . Primers used in this process were designed in Primer Premier 6 (United States), and the primer sequences are given in Total RNA was reverse transcribed into cDNA after residual genomic DNA degradation by ReverTra Ace∘C, 1 ml supernatant of the solution was transferred and evaporated to dryness under nitrogen gas at 37∘C  followed by 1 min vortex and 1 h ultrasound extraction as previously described with minor modifications . After a at 37∘C . The resTM BEH C18 column  was attached to the whole analyses at 35∘C. A flow rate of 0.3 mL/min was chosen to use while 0.1% formic acid water (A) and acetonitrile (B) comprising the mobile phase. The gradient elution conditions were optimized as follows: 0–3 min, 5% B → 5% B; 3–25 min, 5% B → 95% B; 25–28 min, 95% B → 95% B; 28–28.1 min, 95% → 5% B, followed by 4 min re-equilibration.Chromatographic separation was completed on a Shimadzu LC-30AD Series UPLC system  equipped with supplementary SIL-30AC injector and (PAD) SPD-M20A detector. ACQUITY UPLCTM 5600+ system , equipped with an electrospray ionization (ESI) source, was coupled to the UPLC system and used to scan parent ion molecular weight from 100 to 1500. Other MS parameters were set as below: electrospray ionization temperature (∘C): 500 (ESI–); nebulizer gas pressure (psi): 60 (ESI–); ion spray voltage (KV): 4.5 (ESI–); collision energies (V): 35 (ESI–).AB SCIEX TripleTOF2) of the diluting of QC samples in series . Repeatability was evaluated by six replicates of QC samples prepared and extracted parallelly as above. Since all tests are completed in 1 day, precision was investigated by intra-day variability, which was inspected by injecting the QC sample six times along with experimental samples detection in 1 day.To monitor the stability and quality of the non-targeted bioanalysis, the quality control (QC) samples were prepared by pooling 0.2 g powder of each sample and extracted in the same method . Lineari2, PubChem3, NIST4, MassBank5, and METLIN6 databases , including peak recognition , alignment, calibration of the internal standard, filtering and normalization to total area. A three-dimensional data set contained sample information, peak retention time (RT), peak relative intensities and mass-to-charge ratio (m/z) was obtained to perform a series of statistical analysis. PeakView  was recommended to visualize raw data of target components in two-stage mass-to-charge ratio map. Then, based on fragment ion information, such components were identified by comparing to HMDBatabases .de novo assembly in Trinity  unigenes were observed matches in all seven databases . The Nr GO classification indicated that numerous terms were assigned under 25 subcategories of biological process, 21 subcategories of cellular component, and 10 subcategories of molecular function in GO classification . For KOGAll clean reads of each sample were analyzed back to the transcriptome assembly. As listed in Ginkgo leaves at two plant age levels. Parallelly, LO vs. LY and HO vs. HY were selected to evaluate the effect of plant age on flavonoid biosynthesis in Ginkgo leaves at two elevation levels.HY vs. LY and HO vs. LO were used to predict the effect of elevation on flavonoid biosynthesis in Ginkgo trees significantly affected the response of genes expression to elevation change, mainly reflected in the significant increase of down-regulated unigenes.Volcano plots in Ginkgo trees age.As to the influence of plant age, 6,438 DEGs were filtered out from LO vs. LY, 4,915 of which were up-regulated and 1,523 of which were down-regulated. However, only a total of 139 DEGs were found in HO vs. HY, including 79 DEGs up-regulated and 60 DEGs down-regulated. Thereby, there were only nine DEGs observed both in LO vs. LY and HO vs. HY with two up-regulated and two down-regulated synchronously. Obviously, the higher elevation appeared to make gene expression insensitive to the change of Ginkgo trees need more nucleic acids process to cope with the abiotic stress caused by elevation change.Thirty most enriched GO categories of each comparison were plotted in p-values. Moreover, “binding” (GO:0005488) was the most abundant category with 78 DEGs, followed by “cellular process” (GO:0009987) and “metabolic process” (GO:0008152).In LO vs. LY, 45 categories in biological process, 10 categories in cellular component and 49 categories in molecular function were observed significant enrichment. “Catalytic activity” (GO:0003824) was the most enriched category in molecular function, followed by “pyrophosphatase activity” (GO:0016462) and “hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides” (GO:0016818). “Localization” (GO:0051179) was the most enriched category in biological process, followed by “transport” (GO:0006810) and “establishment of localization” (GO:0051234). “Holliday junction helicase complex” (GO:0009379) was the most enriched category in cellular component, followed by “Holliday junction resolvase complex” (GO:0048476) and “DNA helicase complex” (GO:0033202). Further, the up-regulated unigenes were enriched in 120 categories while the down-regulated ones enriched in nine categories. Nevertheless, no categories were significant enriched in HO vs. HY, although “toxic substance binding” (GO:0015643) of molecular function and “immunoglobulin complex” (GO:0019814) of cellular component possessed the lowest corrected All DEGs, as well as the up- and down-regulated DEGs separately, were mapped to the KEGG database to identify genes involved in signal transduction or metabolic pathways. Twenty pathways with the most enrichment degree of each comparison were shown in bar plots.p-value of 3E-4   .p-value between 0.05 and 0.1 , “Alanine, aspartate and glutamate metabolism” (ko00250) and “Glycine, serine and threonine metabolism” (ko00260) were the most enriched pathways with a corrected  and 0.1 . Separat and 0.1  showed t and 0.1 , as well and 0.1 , no one Ginkgo trees of low age showed the disturbance of benzene propane, flavonoid biosynthesis, and circadian rhythm in the face of elevation rise. Flavonoid biosynthesis showed a significant up-regulation at elevated altitude. Ginkgo individuals of different ages at low altitudes show major differences in amino acid synthesis and metabolism. When they became older, significantly up-regulated unigenes are associated with chlorophyll and photosynthesis. Also, fatty acid elongation, phenylpropanoid synthesis, and plant hormone signal transduction were downgraded to varying degrees.In brief, trans-cinnamate 4-monooxygenase genes, 10 caffeoyl-CoA O-methyltransferase genes, 9 chalcone synthase (CHS) genes, 7 chalcone isomerase (CHI) genes, 5 anthocyanidin synthase (ANS) genes, 10 flavonol synthase (FLS) genes, 9 flavonoid 3′-monooxygenase (F3M) genes, 18 anthocyanidin reductase (ANR) genes, 4 coumaroylquinate (coumaroylshikimate) 3′-monooxygenase (C3′H) genes, 16 shikimate O-hydroxycinnamoyltransferase (HCT) genes, 10 leucoanthocyanidin reductase (LAR) genes, 17 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR) genes and 1 flavonoid 3′,5′-hydroxylase (F3′5′H) gene.A total of 136 unigenes were putatively annotated in the flavonoid biosynthesis, including 5 naringenin 3-dioxygenase (F3H) genes, 15 p-value < 0.05 described previously, there were only 11 unigenes observed up- or down-regulation in four comparisons belonging to the flavonoid biosynthesis pathway  ratios were larger than 2 (|log2(FC)| > 1), indicating relatively dramatic variations toward these unigenes. However, in HO vs. LO, there were only one putative DFR unigene up-regulated with a log2(FC) value of 6.72 and one putative FLS unigene down-regulated with a log2(FC) value of −2.08. For LO vs. LY, one putative F3M unigene, three putative DFR unigenes and two putative ANR unigenes were identified different-level up-regulation when the plant age became older. Meanwhile, one putative C3′H unigene, one putative F3H unigene, one putative F3M unigene, one putative DFR unigene and one putative FLS unigene were down-regulated despite several |log2(FC)| values were less than one. Unfortunately, no DEGs were filtered in flavonoid biosynthesis in HO vs. HY.Based on the strict threshold of adjusted  pathway . So, to DFR unigene and one putative FLS unigene were both down-regulated with the increase of elevation. The situation was much more complicated when the influence of plant age was considered in LO vs. LY at a low elevation level. Two putative unigenes of ANR was observed up-regulated, and the filtered putative unigenes of C3′H, F3H, and FLS were all down-regulated with the decrease of plant age. Additionally, F3M possessed one up-regulated and one down-regulated putative unigenes. For DFR, three putative unigenes were up-regulated and one was down-regulated as plant age increased. Unexpectedly, no unigenes related to flavonoid biosynthesis were found significant variation when elevation increased in HO vs. HY. Seemingly, the flavonoid biosynthesis pathway was more sensitive and easier to be influenced when wild Ginkgo individuals were at low elevation or young age level. DFRs may be the key regulatory point for flavonoid biosynthesis disturbance in Ginkgo leaves.In summary, a higher elevation led to more unigenes up-regulated than down-regulated in flavonoid biosynthesis in HY vs. LY at a young plant age level. However, when plant age became older in HO vs. LO, only one putative 2(FC) results  of six QC samples obtained from LC–MS in negative mode  demonstrGinkgo trees grow in the wild.According to the non-targeted metabolites detection and identification by UPLC/qTOF MS method, A total of 13 ingredients involved in flavonoid biosynthesis pathway and 11 flavonoid downstream derivatives that possessed high parent ion signal intensity in MS were predicted and confirmed based on fragment information in multiple databases. The details of these ingredients, including RT, theoretical and experimental m/z, mass error and ions fragment information, were summarized in O-neohesperidoside, isorhamnetin-3-O-rutinoside, kaempferol 3-O-β-D-rhamninoside, quercetin-3-neohesperidoside-7-rhamnoside, rhamnegin, quercitrin and quercetin 3-β-D-glucoside) were seemingly more abundant both in HY than in LY and in HO than in LO, which indicated that the increase of elevation may likely lead to the rise of the total flavonoid content. Simultaneously, 17 ingredients -catechin, (+)-gallocatechin, (−)-epicatechin, kaempferol, quercetin, myricetin, chrysin, apigenin, luteolin, rutin, isoginkgetin, ginkgetin, kaempferol-7-O-neohesperidoside, isorhamnetin-3-O-rutinoside) were more abundant both in LY than in LO and in HY than in HO. What’ more, a similar trend was observed in the comparison HO vs. HY about kaempferol 3-O-β-D-rhamninoside, quercetin-3-neohesperidoside-7-rhamnoside, rhamnegin, quercitrin and quercetin 3-β-D-glucoside. These signs suggested that an increase in plant age was more likely to result in a decrease in total flavonoids.Overall, 10 ingredients seemingly showed higher concentration in LY than that in other three groups, including naringenin chalcone, pinocembrin, dihydrokaempferol, (+)-gallocatechin, (−)-epicatechin, kaempferol, quercetin, chrysin isoginkgetin and ginkgetin, although all fold changes were not large. Dihydrokaempferol was significantly less abundant in HY than that in LY. Dihydroquercetin and chrysin were both significantly less affluent in HO than LO. Irrespective of significance, the average contents of apigenin and luteolin were likely richer in HY than that in LY. Apart from isoginkgetin and ginkgetin, all other flavonoid downstream derivatives , cinnamyl-alcohol dehydrogenase (CAD), peroxidase (PRD), beta-glucosidase (bGLD), and pinoresinol/lariciresinol reductase (PLR) were observed visibly variability of gene expression in different comparisons , C4H, C3H, COMT, and F5H were the key enzyme in the process of converting cinnamic acid into HCAs. One putative C4H unigene and five putative COMT unigenes were observed different levels of fluctuation. However, the concentration statistics of p-Coumaric acid, Caffeic acid, Ferulic acid, and Sinapic acid indicated no significant variation between four comparisons. Seven putative bGLD unigenes were screened with notable differential gene expressions, which indicated that the rise of elevation might promote the conversion from cis-beta-D-Glucosyl-2-hydroxycinnamate to cis-2-Hydroxycinnamate while an older plant age suggesting a negative regulation on this process. Nevertheless, related metabolite detection is not significant enough to support this conclusion.CAD unigenes and five down-regulated PRD unigenes may be the main cause of a marked accumulation of p-Coumaryl alcohol when the elevation rose in HY vs. LY. They also have the potential possibility to promote the production of alcohols such as Caffeyl alcohol, Coniferyl alcohol, etc., although these metabolites not been identified. In this case, the up-regulation of CADs and the down-regulation of PRDs may lead to the inhibition of lignins  biosynthesis, which is more conducive to the synthesis of lignans. However, there was no significant evidence of PLRs and SIRDs, as well as the only concentration analysis of Secoisolariciresinol to support this speculation. When the age of Ginkgo trees increased in LO vs. LY, although some putative unigenes of CAD, PRD, and PLR showed an upward trend, the similarity of functional annotations was not high. In addition, there was no indication that the concentrations of p-Coumaryl and Secoisolariciresinol have increased significantly.Noteworthily, five up-regulated CADs and down-regulating PRDs. Thus, a hypothesis was presumed that the rise of elevation might potentially inhibit the synthesis of lignins, leading to an accumulation of Caffeyl alcohol, Coniferyl alcohol, etc. HCAs and lignans showed no obvious variational trend in this situation. Age variability caused related genetic perturbations, but there was insufficient evidence to prove the specific impact.Hence, the elevation increase may lead to the accumulation of lignin monomers, like 4-Coumaryl alcohol, Coniferyl alcohol, 5-Hydroxyconiferyl alcohol and Sinapyl alcohol, but inhibit the lignification by up-regulating Flavonoids have long been recognized as the primary antioxidants in plants which are useful in preventing UV-B radiation from penetrating into sensitive tissues and protecting against programmed cell death mediated by oxidative stress . The risDFR unigenes existed in multiple plant species -epicatechin) has been reported to be simultaneous with the age increase of Cistus clusii, a perennial evergreen bush (Ginkgo trees (LO vs. LY in DFRs and ANRs (Ginkgo age (LO vs. LY). On the other hand, although the senescence process has been reported to be correlated with the accumulation of phenolics in Pinus needles (Ginkgo senescence. Therefore, whether flavonoids play a vital role in the senescence of Gingko individuals requires further research.Nowadays, the temporal effects on the flavonoid biosynthesis were mainly focused on seasonality, circadian rhythm and the growth stages of leaves, flowers or fruits in herbs , whilst een bush . Howeverand ANRs , the rel needles , no simiLigustrum vulgare leaves (Arabidopsis (There was no significant correlation between HCAs and elevation increase in this study. Increased altitude means increased illumination. The HCAs changes under light induction have long been questioned. Excessive sunlight exposure led to a decrease of HCAs:flavonoid ratio in e leaves , which hbidopsis . Howeverbidopsis . DiffereArabidopsis seedlings (Ginkgo leaves, no significant signs were observed to reflect the light influence on lignin synthesis. The light-induced enhancement of lignin biosynthesis may be not fully applicable to the leaves of mature woody plants. Lignin biosynthesis genes are preferentially expressed in actively lignifying tissues, while many lignin repressors are preferentially expressed in non-lignifying tissues (Ginkgo individuals, the lignification degree of the trunk and branches always are much higher than that of the leaves, so the expression of the lignification gene should be mainly concentrated in the branches rather than the leaves. This may also be the potential reason why the age variability of Ginkgo trees had not caused significant changes in the lignin synthesis pathway in leaves in this study.Lignin biosynthesis, as a highly energy-consuming and irreversible process, responds to many environmental factors like light, sugar content, biological clocks . It has eedlings  and hexoeedlings . However tissues . For matForsythia koreana and Sesamum indicum, and verifications of more species need to proceed further.The illumination change caused by elevation rise showed insignificant disturbances for lignans biosynthesis in this study. CPI-FK cells were reported to show a positive response to white fluorescent, red LED and blue LED light in the synthesis of lignans, indicating that light enhancement has the potential to improve lignans production . HoweverIn summary, the weak illumination enhancement caused by the elevation change may be the main reason for the significant accumulation of lignin monomers. However, there were no significant changes in HCAs, lignins and lignans. The potential reason was the mild variability of environmental factors, unlike the artificial designs of dramatic environmental difference. In addition, different physiological status caused by age changes did not cause significant fluctuations in the biosynthesis of non-flavonoids polyphenolics. As few insights were thrown to study the influence of individual age on its tissue metabolism, more focus and discussions should be paid to this field.Arabidopsis roots appeared to support this inference (An elevation increase of 300–400 m may result in a moderate enhancement in illumination intensity. In this situation, DEGs analysis seemed to show a stronger disturbance in flavonoid biosynthesis than in other non-flavonoids polyphenolics pathways, which indicated that flavonoid biosynthesis might be more sensitive to elevation and tree age than other branch pathways involved in phenylpropanoid biosynthesis. Metabolites profile seemed to support this conjecture. Otherwise, the synthesis of these polyphenolics is based on the same substrate, which will inevitably lead to the existence of competition. A light-induced metabolism analysis in nference .Ginkgo trees, based on transcriptomics and metabolomics analysis. The key genes involved in flavonoid biosynthesis, including DFR, LAR, and ANR were significantly up-regulated with the elevation increase. Mass spectrometry analysis indicated a similar potential trend that the content of most flavonoid derivatives increased with the elevation rise. The light fluctuation caused by elevation increase may be the main impetus in this process. DFRs appeared to exhibit the most sensitive responses in the face of environmental changes; therefore, might be the main regulatory point in the flavonoid synthesis. Although the age variability only affected the expression of related genes to a lesser extent, metabolites analysis speculated that the growth of Ginkgo age might be detrimental to flavonoid synthesis in leaves. In addition, multiple genes implicated in the synthesis of HCAs, lignin, and lignan exhibited fluctuations with the elevation changes. Notably, significantly up-regulated CADs and down-regulated PRDs potentially led to the accumulation of p-Coumaryl alcohol, one of the lignin monomers, and might inhibit further lignification, while the age increase did not observe a significant impact. Therefore, flavonoid biosynthesis exhibited a higher degree of perturbation than that of HCAs, lignins and lignans, potentially suggesting that flavonoid biosynthesis might be more susceptible than other branch pathways involved in phenylpropanoid biosynthesis. However, moderate environmental changes may mask fluctuations in insensitive components in this study. And the effect of Ginkgo age on the synthesis of phenylpropanoids in leaves needs further excavation. In conclusion, these findings can facilitate the functional genomic and metabolic research in Ginkgo, and provide a reference for artificial planting and industrial harvesting.This research is the first insight into the effects of elevation and plant age on phenylpropanoid biosynthesis in wild KZ, YL, QY, and XL conceived and designed the work. KZ, QY, SF, WC, and RL performed the experiments. KZ, DZ, WC, and RL analyzed the data. KZ wrote and revised the manuscript. All authors read and approved the final manuscript.WC was employed by the company Zhejiang CONBA Pharmaceutical, Co., Ltd., Hangzhou, China. The remaining 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.


Mycoplasma hyopneumoniae infections are responsible for significant economic losses in the swine industry. Commercially available vaccines are not able to inhibit the colonisation of the respiratory tract by M. hyopneumoniae absolutely, therefore vaccination can be completed with antibiotic treatment to moderate clinical signs and improve performances of the animals. Antibiotic susceptibility testing of M. hyopneumoniae is time-consuming and complicated; therefore, it is not accomplished routinely. The aim of this study was to determine the in vitro susceptibility to 15 different antibiotics of M. hyopneumoniae isolates originating from Hungarian slaughterhouses and to examine single-nucleotide polymorphisms (SNPs) in genes affecting susceptibility to antimicrobials. Minimum inhibitory concentration (MIC) values of the examined antibiotics against 44 M. hyopneumoniae strains were determined by microbroth dilution method. While all of the tested antibiotics were effective against the majority of the studied strains, high MIC values of fluoroquinolones  were observed against one strain (MycSu17) and extremely high MIC values of macrolides and lincomycin  were determined against another, outlier strain (MycSu18). Amino acid changes in the genes gyrA  and parC  correlated with decreased antibiotic susceptibility to fluoroquinolones and a SNP in the nucleotide sequence of the 23S rRNA (A2059G) was found to be associated with increased MIC values of macrolides. The correlation was more remarkable when final MIC values were evaluated. This study presented the antibiotic susceptibility profiles of M. hyopneumoniae strains circulating in the Central European region, demonstrating the high in vitro efficacy of the tested agents. The observed high MIC values correlated with the SNPs in the examined regions and support the relevance of susceptibility testing and directed antibiotic therapy. Mycoplasma hyopneumoniae is a member of the class Mollicutes [M. hyopneumoniae infection affects especially growing pigs, causing significant economic losses in the swine industry worldwide, by chronic cough, growth retardation and predisposing animals to secondary infections [llicutes  and the llicutes . M. hyopfections –6. Improfections ,7,8. As fections , antibiofections .Mycoplasmas are resistant to antimicrobials that interfere with folic acid metabolism and cell wall synthesis, like sulphonamides, trimetoprim and the β-lactam class of antibiotics [M. hyopneumoniae [M. hyopneumoniae to fluoroquinolones, macrolides, lincosamides and tetracyclines [in vitro susceptibility testing with microbroth dilution method were laid down almost 20 years ago [ibiotics ,10. Macreumoniae ,11. Howecyclines –14. The cyclines ,15,16. Tears ago , howeverM. hyopneumoniae infection in Hungary [gyrA, gyrB, parC, parE) [parC gene  [gyrA gene was also described to be related to decreased susceptibility to enrofloxacin in M. hyopneumoniae [gyrA gene observed in other Mycoplasma species were for example Gly81Ala or Glu87Gly [M. hyopneumoniae due to a G2057A transition in the 23S rRNA sequence [Genomic changes (e.g. single-nucleotide polymorphism (SNP)) related to decreased effectiveness of certain antibiotics have been identified in previous publications ,15,18,19 Hungary ,21. The C, parE) . The majGlu87Gly ,24. Accosequence . In addisequence ,25.M. hyopneumoniae strains isolated from Hungarian slaughterhouses in years 2015 and 2016, against 15 antimicrobial agents and to examine the genetic background of increased MIC values.The aim of this study was to describe the antibiotic susceptibility profile of 44 M. hyopneumoniae strains originating mainly from Hungary (n = 40), but also from Slovakia (n = 3) and the Czech Republic (n = 1) were tested in this study  according to the manufacturer’s instructions. Species-specific PCR test was accomplished to confirm the presence of M. hyopneumoniae [Mycoplasma species sequence analyses and BLAST search were carried out using the amplicons of a universal Mycoplasmatales PCR system targeting the 16S/23S rRNA intergenic spacer region [Forty-four is study  Tables. eumoniae . To exclr region . PCR pro4−105 CCU/ml of the strains as recommended by Hannan [50 and MIC90 values were determined as the lowest concentrations that inhibited the growth of 50% or 90% of the strains [The number of colour changing units (CCU) was determined by microbroth dilution method after four weeks of incubation . Antimicy Hannan . All str strains .M. hyopneumoniae were examined in genes gyrA, gyrB, parC, parE and 23S rRNA [gyrA and gyrB primers and heat profile were used according to Vicca et al. [parC and parE, primers and heat profile were used according to Le Carrou et al. [et al. [5’ GAT GAG TAT TCT AAG GTG AGC GAG 3’) and reverse (5’ CAG TCA AAC TAC CCA CCA CG 3’) primers. PCR products were sequenced on ABI 3130XL genetic analyser  and sequence analysis was performed by using Geneious software 10.2.3 (Biomatters Ltd.) [Escherichia coli strain K-12 substrain MG1655 (GenBank accession number CP014225). Susceptibility profiles and correlating genetic markers were evaluated in relation with previously determined genotypes of the examined strains also [Genetic markers correlating with antibiotic susceptibility in 23S rRNA ,15,18,19 [et al.  were use3 difference between MIC values) than that reported in the literature (≤0.001–0.002 μg/ml) [50 and MIC90 values of each antibiotic against the examined strains, are recorded in The initial MIC values are evaluated and discussed throughout the study , however2 μg/ml) . PreviouM. hyopneumoniae are not standardized, MIC values were compared to previously published, unofficial breakpoints [As official breakpoints of antibiotics against akpoints  in the pakpoints .50 value at the lowest antibiotic concentration (≤0.039 μg/ml), while the other values represented equipartition with the highest MIC values   was observed in the MIC values of tylosin and tylvalosin against the examined strains. MIC50 values of gamithromycin and tulathromycin were 0.5 μg/ml, while MIC90 values were 2 μg/ml and 1 μg/ml, respectively. Both MIC50 and MIC90 values of lincomycin coincided with the lowest examined antibiotic concentration (≤0.25 μg/ml)  showed one main peak coinciding with MICctively) . One strycycline . The lowycycline . MIC50 aed value . Five maed value , out of 5 μg/ml) . For all5 μg/ml) . The MIC 2 μg/ml .gyrA, gyrB, parC and parE); however, only SNPs resulting in amino acid alterations were further examined in the present study. None of the amino acid changes in the genes gyrB and parE showed correlation with the defined MIC values. On the other hand, amino acid changes in the gyrA gene  and in the parC gene  correlated with decreased susceptibility of fluoroquinolones . It is noteworthy, that the double substitutions in strain MycSu44 consisted of Ala83Val in gene gyrA and Asp84Asn in parC, while the rest of the strains showed various amino acid substitution types in gene gyrA but only the change of serine at amino acid position 80 in gene parC. The one outlier strain (MycSu17) against which 2.5–5 μg/ml initial MIC values of fluoroquinolones were detected contained the double substitution combination Ser80Phe (in parC gene) with Ala83Val (in gyrA gene). Correlation was described between increased MIC values of macrolides and lincosamides against Mycoplasma species/ M. hyopneumoniae and SNPs in the 23S rRNA sequence [High MIC values of fluoroquinolones, macrolides and lincosamides, exceeding the unofficial breakpoints  were fouinolones . Single sequence ,19. A nusequence  showed dAntibiotic susceptibility testing of porcine mycoplasmas is not performed routinely, because it is fastidious, time-consuming and requires special techniques and media . FurtherM. hyopneumoniae through inhibition of the bacterial DNA gyrase and topoisomerase IV enzymes [50 value of enrofloxacin, similarly to previous results in other European publications in the last 20 years [et al. [50 value of marbofloxacin against the studied strains was mostly in accordance with recent data, MIC90 value against the Hungarian isolates was higher than those against Belgian, Spanish and British strains (0.5–1 μg/ml) [Fluoroquinolones are potentially active antimicrobial agents against  enzymes ,22. In t20 years ,13,34. O [et al. . Similar [et al. , which fgyrB and parE, corroborating earlier publications [parC gene  showed correlation with increased MIC values of fluoroquinolones in earlier publications [parC and gyrA genes were described in the examined strains. The observed effect of the double substitutions is in accordance with previous findings of Vicca et al. [gyrA and parC, defining a unique combination in the outlier strain (MycSu17). Moreover, new amino acid alterations (Glu87Gly and Gly81Ala) have been described in gyrA gene of M. hyopenumoniae in the present study, which had been observed only in M. bovis and M. gallisepticum before [No amino acid substitutions, correlating with increased MIC values, were observed in the genes ications . Althougications ,18,19, tm before ,24. Factm before , correlamax/MIC ratio) of equal or higher than 10 was proposed [max in pigs [The increasing susceptibility against fluoroquinolones is a notable problem, because these agents are important antibiotics for human therapy . To maxiproposed . Marbofl in pigs  respectiM. hyopneumoniae infections, and they act by binding to the decoding centre of the small ribosomal subunit of the bacterium [50 and MIC90 values of oxytetracycline [50 and MIC90 values of doxycycline were described against strains originating from Spain (1 μg/ml both) and Thailand (3.12 μg/ml and 6.25 μg/ml) than against the Hungarian isolates. According to other publications supported also by our results, tetracyclines are still active against M. hyopneumoniae despite of their long-standing usage in human and veterinary medicine [Tetracyclines are frequently used to control acterium ,41. Mostacycline ,13,34; bmedicine ,33.M. hyopneumoniae, as low MIC50 and MIC90 values were observed in the present study, similarly to earlier data [50 and MIC90 values were higher in the present study compared to Spanish and Belgian MIC values [The aminoglycoside gentamicin seems to be an effective antimicrobial agent against ier data ,32. AlthC values ,32.M. hyopneumoniae infections [50 and MIC90 values of macrolides was described compared to the literature [E. coli numbering), which was also described in macrolide and lincosamide resistant M. bovis strains before [et al. [Macrolides are among the most frequently used antibiotics in the swine industry to treat fections . Both 16fections . The reafections ) in eithterature , and extterature ,19,43. As before . Accordi [et al.  and the  [et al. , Thailan [et al.  and Spai [et al. .M. hyopneumoniae, but extremely high MIC values appear every now and then [Lincomycin is also active against and then ,32,33, land then . The simand then .M. hyopneumoniae infections through inhibiting bacterial protein synthesis [M. hyopneumoniae with low in vitro inhibitory concentrations [Pleuromutilins are important antibiotics to control ynthesis . Accorditrations –13,32–34trations ,32,34.50 and MIC90 values of florfenicol, were similar to earlier observations from different parts of Europe and Thailand [M. hyopneumoniae.The chloramphenicol derivative florfenicol is an inhibitor of bacterial protein synthesis, used exclusively for veterinary purposes . The modThailand ,33,34, aIn vitro MIC values do not necessarily correlate with the effectiveness of the antimicrobials in vivo and interpretation of the MIC distributions is difficult as Mycoplasma species with veterinary relevance do not have official clinical breakpoints [in vivo antimicrobial activity [M. hyopneumoniae. Although the isolation of M. hyopneumoniae strains is a time-consuming and fastidious process, the regularly accomplished antibiotic susceptibility testing of the swine herds should enable appropriate antibiotic usage during treatment. Furthermore, the development of PCR-based susceptibility tests based on SNPs correlating with changes in the MIC values, could improve diagnostics and treatment, similarly to antibiotic susceptibility testing in M. bovis [akpoints . Furtherakpoints . PK/PD  or macrolides and lincomycin (MycSu18) were observed. Single or double amino acid substitutions in the genes gyrA , parC  and a SNP in the 23S rRNA sequence (A2059G) were also detected correlating with decreased antibiotic susceptibilities. Macrolides and fluoroquinolones are frequently used empirically as a first choice for the management of mycoplasmoses in livestock in Europe. The regular testing of the sensitivity profile of M. hyopneumoniae, the determination of herd specific MICs would promote the use of less critical antibacterials , and might contribute to the preservation of the critically important antibiotics (macrolides and fluoroquinolones) both for veterinary and human medicine.This study provided current and relevant information about the antibiotic susceptibility profiles of S1 TableIsolation data  and MIC values of enrofloxacin (EFX), marbofloxacin (MFX), oxytetracycline (OTC), doxycycline (DX), gentamicin (GTC), spectinomycin (SPC), tylosin (TYL), tilmicosin (TIL), tylvalosin (TVN), gamithromycin (GTM), tulathromycin (TTM), tiamulin (TIA), valnemulin (VAL), lincomycin (LCM) and florfenicol (FFC) are presented. Abbreviations for herd of origin are: H-Hungary, CZ-Czech Republic, SK-Slovakia.(DOCX)Click here for additional data file.S2 TableIsolation data  and MIC values of enrofloxacin (EFX), marbofloxacin (MFX), oxytetracycline (OTC), doxycycline (DX), gentamicin (GTC), spectinomycin (SPC), tylosin (TYL), tilmicosin (TIL), tylvalosin (TVN), gamithromycin (GTM), tulathromycin (TTM), tiamulin (TIA), valnemulin (VAL), lincomycin (LCM) and florfenicol (FFC) are presented. Abbreviations for herd of origin are: H-Hungary, CZ-Czech Republic, SK-Slovakia.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Introduction: The intracerebral hemorrhage (ICH)-score is used for estimation of patients' prognosis. The hemorrhage volume calculated from computed tomography (CT) contributes as one main factor. Several studies have proven that dimensions of an ICH may be displayed sufficiently by transcranial sonography (TCS). Yet, the adequacy of ICH-volumetry via TCS in calculating the ICH-score and its use as prognostic tool has not been studied.Methods: Forty consecutive patients with supratentorial ICH diagnosed via CT were included in this prospective observational pilot study. 45 examination-series via CT and TCS were done in order to perform an ICH-volumetry and calculate the ICH-score. Volume was calculated using the ABC/2 estimation. Results of both imaging techniques were compared regarding quantification of ICH- volume and correct prognostication. A modified Rankin Scale (mRS)-score of 0–3 points was valued as good outcome.Results: The imaging techniques did not show a difference in volumetry (p = 0.794) and TCS derived hemorrhage volume correlated significantly with ICH-volume measured on CT-scans. Calculated ICH-scores also did not differ (p = 0.323). Patients with an ICH-score larger than 2 points were predicted to experience a poor outcome at discharge with mRS 4–6 points, and the prognostication of the outcome was correct. Patients with a good outcome showed a smaller ICH-volume (11.2 ± 9.1ml) than patients with a poor outcome .Conclusion: Volumetry in supratentorial ICH via TCS is feasible and the prognostication with the ICH-score based on its results is comparable to CT-imaging and sufficient. Intracerebral hemorrhages (ICH) account for 10–15% of all strokes and are associated with a poor prognosis. Although a relevant portion of patients receives maximal care, the overall mortality rate reaches up to 45% , 3. The Cerebral structures and pathologies can be visualized via transcranial gray-scale sonography (TCS) with a high sensitivity and specificity comparable to CT-scans. TCS also allows monitoring of ICHs and their resorption course . So far,Forty consecutive patients with supratentorial ICH who were admitted to the Neurological Intensive Care Unit (NICU) of the University Medical Center in Freiburg were included into this prospective observational pilot study. ICH was diagnosed via CT-scans on admission by a neuroradiologist. The ICH-score was calculated based on clinical and imaging data as described previously , 3. FurtTCS was done by experienced neurologists on admission. It was performed using a GE Logique 7 expert ultrasound system  with a 2.0–2.5 MHz-transducer on the transtemporal approach in a meato-orbital plane. Visualization of hyperechogenic blood was performed progressing from the contralateral side of the lesion to a scanning-depth of 16 cm and tilting the probe to the maximum extent of the hemorrhage. So, first its length (A) was measured, followed by measuring the width (B) in a 90 degree-angle. Third, the dimension of the height (C) of the ICH was measured in the coronal sectional plane. ICH-volume was calculated by multiplying length (A), width (B) and height (C) divided by 2  4, 6). , 6. 4, 6Localization of ICH was described, and ICH-volumetry done on CT-scans with a slice-thickness of 5 mm analogously to TCS with ABC/2 by an experienced stroke neurologist blinded for TCS-data.After testing for normal distribution clinical, TCS- and CT-data was compared descriptively and correlated via non-parametric statistics. TCS and CT derived ICH-scores were compared using non-parametric testing. Bland-Altman-analyses of TCS- and CT-volumetry were calculated; a maximum of a 10%-variance of volumes measured in comparison to CT would confirm validity of TCS . The firForty patients diagnosed with ICH were included in this study. Patient characteristics are summarized in p = 0.748). Volumes showed a strong linear correlation between TCS- and CT- ICH volumes  on CT-scans and 18.7 ml (9.3–30.0) on TCS did not differ . Patients with an ICH-score > 2 were predicted to experience a poor outcome (mRS 4–6) at discharge. The predicted outcome did not differ from the actual outcome at discharge .Calculating the ICH-score with the CT-data revealed a median score of 1.0 (IQR 1.0–2.0) which did not differ from the TCS-ICH-score with a median of 1.0  and 26 had a poor outcome (mRS 4–6).p = 0.002; p = 0.013; 2(6) = 13.491, p = 0.036]. We could not detect a difference of ICH-volumes in the 35 patients surviving the hospital stay and the 5 patients who deceased (p = 0.277).Patients with a good outcome had significantly smaller ICH-volumes based on TCS-examination compared to patients with a poor outcome [good: mean volumes 11.2 (±9.1) ml vs. poor: 38.2 (±41.2) ml; The results of this prospective pilot study show that TCS-volumetry using the GE Logique 7 expert ultrasound system in supratentorial ICH is highly comparable to CT-data and therefore yields the same ICH-scores. ICH-volumes differed regarding outcome reflected by mRS-levels at discharge, and prognostication regarding a good or poor outcome was correct based on TCS volume-measurements and the ICH-score calculated from it.Among the existing prognostic scores, the ICH-score is the most widely used in clinical practice. The ICH-score is a simple prognostic-tool. It comprises the GCS-score defining the severity of clinical symptoms, additionally modified by age, ICH-volume and –location , 3. A 30It has been suggested to use surgical methods based on the ICH-score to reduce mortality in ICH-volumes greater than 30ml and GCS-scores below 12. ICH-Volume seems to be the strongest factor influencing patients' outcome , 9. The We were able to examine all patients with TCS. Difference between TCS and CT-volumetry was very low in all patients. It is often pointed out that this technique is limited by insufficient transtemporal bone window (in 16–36% of the European population) . In geneWe conclude that calculating ICH-volume via TCS is feasible and that prognostication with the ICH-score based on its results is comparable to using CT-imaging.This study was carried out in accordance with the recommendations of the local ethic committee of Freiburg and the Declaration of Helsinki. The local ethic committee of Freiburg approved the protocol (EK 28/10). As this is an observational study, the board waived the need for written patients' consent.HF did the literature research, performed the statistics and wrote the first draft of the manuscript. W-DN contributed conception. W-DN and JB designed the study. W-DN, AS, and JB gathered clinical, sonographic and radiological data. W-DN, JB, and AS contributed sections to the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.AS received travel funding from Ipsen and speaking honoraria from Bayer Health Care. JB received speaking honoraria from Bayer Health Care, Boehringer Ingelheim, Daiichi Sankyo and Pfizer. The remaining 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.


Transfer of genetic material from microbes or viruses into the host genome is known as horizontal gene transfer (HGT). The integration of viruses into the human genome is associated with multiple cancers, and these can now be detected using next-generation sequencing methods such as whole genome sequencing and RNA-sequencing.We designed a novel computational workflow, HGT-ID, to identify the integration of viruses into the human genome using the sequencing data. The HGT-ID workflow primarily follows a four-step procedure: i) pre-processing of unaligned reads, ii) virus detection using subtraction approach, iii) identification of virus integration site using discordant and soft-clipped reads and iv) HGT candidates prioritization through a scoring function. Annotation and visualization of the events, as well as primer design for experimental validation, are also provided in the final report. We evaluated the tool performance with the well-understood cervical cancer samples. The HGT-ID workflow accurately detected known human papillomavirus (HPV) integration sites with high sensitivity and specificity compared to previous HGT methods. We applied HGT-ID to The Cancer Genome Atlas (TCGA) whole-genome sequencing data (WGS) from liver tumor-normal pairs. Multiple hepatitis B virus (HBV) integration sites were identified in TCGA liver samples and confirmed by HGT-ID using the RNA-Seq data from the matched liver pairs. This shows the applicability of the method in both the data types and cross-validation of the HGT events in liver samples. We also processed 220 breast tumor WGS data through the workflow; however, there were no HGT events detected in those samples.http://kalarikrlab.org/Software/HGT-ID.html.HGT-ID is a novel computational workflow to detect the integration of viruses in the human genome using the sequencing data. It is fast and accurate with functions such as prioritization, annotation, visualization and primer design for future validation of HGTs. The HGT-ID workflow is released under the MIT License and available at  Streptococcus pneumoniae cells into infectious cells by exposing them to an extract made from virulent but dead S. pneumoniae cells [MYC oncogene that is responsible for this disease [Horizontal gene transfer (HGT), or the transfer of genes between organisms in a manner other than traditional reproduction, was first described in 1928 when Frederick Griffith converted nonvirulent ae cells . With thae cells , 5. Althae cells , some caae cells –9. Theseae cells . For exaae cells . Also, rae cells . Similarae cells –13; hereae cells , presumaae cells . Merkel ae cells , 16. Herae cells . Finally disease , 19.Increasingly, researchers have been interrogating RNA-Seq data to determine whether the expression of viral sequences is associated with other types of cancer as well. Two recent studies have attempted to identify viral signatures in RNA sequencing data from many different types of cancers , 21. TheExisting methods for identifying viral integration sites are based on the subtraction approach, which removes mapped human reads and focuses on unmapped reads in the aligned bam files. For example, the VirusSeq software  was one HGT-ID follows a four-step procedure that includes the preprocessing of a previously aligned BAM file to the human genome, the detection of viral species with unmapped reads, identification of the viral integration sites as HGT candidates, and finally the priority score assignment by a scoring function Fig. .Fig. 1OvAs input, HGT-ID requires paired-end next-generation sequencing (NGS) data in the standard BAM file format generated by any aligner using the human genome reference. Unmapped reads from the BAM file are extracted and then remapped to the human reference genome (hg19) using BWA-mem  to removftp://ftp.ncbi.nih.gov/refseq/release/viral, as of March 2015) and is a reasonable collection of representative consensus sequences for different strains. Potential viral reads from the preprocessing step above are then aligned to the RefSeq viral reference genome using the BWA-mem software. After the viral alignment, read pairs with both ends mapped to viral species only are filtered. As direct evidence of viral integration, reads with one end mapped to the viral genome and other end assigned to the human genome are retained for further analysis. In order to remove low complexity sequence that is common in viral sequences and might affect the alignment, we calculate the sequence linguistic complexity (LC) score [p-value less than 0.01 for each kept read. The remaining discordant read pairs are considered as confident supporting reads for the viral integration step. Although we have set the default to recommended values, all the parameters listed in this section are customizable through the configuration files by the user.For the viral detection, we use the RefSeq Viral genome database  as the rC) score  of each C) score . Reads wThe viral integration sites are identified in a two-step process. First, for the discordant read pairs, HGT-ID clusters the human reads by their genomic location. The clusters then expand to both upstream and downstream directions recursively  until no more human reads from discordant read pairs can be recruited. For each cluster, a putative breakpoint is then estimated by taking the average of the start points of all reads in the cluster. The same procedure is also applied to the virus side to obtain a putative viral genomic breakpoint Fig. .Fig. 2DiIn the second step, HGT-ID scans for soft-clipped human reads around the putative breakpoint. The search window is centered at the breakpoint, spanning both upstream and downstream regions to match the size of the library fragments. Before each soft-clip read can be recruited into the read cluster, the soft clipped section is compared with the viral genome to remove spurious soft-clipped reads that do not belong to the virus. Among the cleaned reads, if there are soft-clipped reads that span through the breakpoint, a precise integration site can be inferred for the human side Fig. . OtherwiThe goal of HGT-ID is to identify high confident HGT events that are associated with high genomic instability. High confident HGT events tend to have high read coverage that supports the event against the background. On the other hand, false positive HGT events are indicative of a relatively low number of supporting reads that might occur due to random chimeric integration of fragments during sequencing . Thus, tNlocal is counted by including all the reads falling in a window that is centered at the breakpoint and spanning both upstream and downstream for the library fragment length. The local probability of a human read to randomly integrate with viral reads can be roughly estimated as PH = mH/Nlocal, where mH is the number of human reads that are either split or spanning through the breakpoints. Similarly, for the integrated viral reads, we can calculate PV = mV/Nlocal, where mV is the number of viral reads that are either split or spanning through the breakpoints. Then, the probability of supporting coverage generated by a random integration of human and viral reads should be proportional to the product of PH and PV. The expected number of random discordant reads countbg can then be estimated as:To estimate the local expected background for a given candidate event, first, the local coverage countsp) is calculated as the sum of discordant read pairs (countD), soft-clipped reads identified in human (countsch) and viral (countscv) bam files respectively, i.e.,The supporting coverage of the given candidate event  using Primer3 [The HGT candidates can be typically validated by polymerase chain reaction (PCR) experiments. The HGT-ID workflow thus provides a primers design function, which designs oligonucleotide primers that flank the detected viral integration sites . Beyond the details of each candidate event and the designed primers, the report also gives circos plots to visualize the location and coverage of each event in both human genome and viral genome.For each sample processed through the workflow, the method provides a comprehensive report in HTML with annotation, visualization and customer primer design for experimental validation  from the actual inserted site, it was counted as true positive.Table http://kalarikrlab.org/Software/HGT-ID.html) of HGT events indicated that the optimal cutoff scores across different coverages was 0. It is noted that the performance evaluations of HGT-ID were based on this cutoff if not otherwise stated.From the performance evaluation in Table Different color lines illustrate different coverages. The false positive ratio (FPR) was calculated as the ratio of the number of false positives and the number of total identified HGT events. The true positive rate (TPR) was calculated as the ratio of the number of true positives and the number of total positives. The coverages were down-sampled from 40× to 30X, 20X, 10X, 4X and 2X, respectively.http://kalarikrlab.org/Software/HGT-ID.html).We applied the HGT-ID workflow to a publicly available WGS dataset (SRA180295) with at least 30× coverage containing four HPV-positive samples: two HPV-positive cell lines (SiHa and HeLa) and two cervical carcinomas (T4931 and T6050)  . The four missing events have no discordant human-viral read pairs, resulting in their being filtered out from our candidate events. Further investigation of the missing events revealed that these four events consisted of very short viral insertions (~ 60 bp) that were smaller than the read length (90 bp). Thus, there were no complete viral reads to form a discordant pair to pass the minimal evidence required for an HGT candidate event in HGT-ID.We compared our results with the original paper, which provided experimental validation for 22 randomly selected viral integration sites from 13 tumor samples. HGT successfully identified 18 of these 22 experimentally identified viral integration sites, with all 18 scoring 10 or higher  from the same data set, which contained false positive HGT events that the original publication identified as candidates but failed to validate. HGT-ID did not pick up any negative events reported in these five samples. While this did not indicate that all other candidate events identified by HGT-ID were true positives due to the limited validation available, HGT-ID had exhibited great performance in accuracy. Overall HGT-ID accurately identified and confirmed 23/27 events (85.2%). On the contrary, VirusFinder 2.0 identified only 16 of 22 (72.7%) [http://kalarikrlab.org/Software/HGT-ID.html). The HGT algorithm was applied to all of the samples with the default settings, and integration events with a score > 10 were reported for both DNA and RNA samples.To check the performance of HGT-ID in both DNA and RNA sequencing data, we processed paired WGS (100 bp PE) and RNA-Seq samples (50 bp PE) from seven TCGA hepatocellular carcinoma (HCC) samples that were originally contributed by the Mayo Clinic. The summary of NGS reads for WGS and RNA-Seq platforms for these seven tumor-normal pairs are described in the website . In addition, HGT-ID workflow identified zero HGT events and a total of 42 HGT candidates in liver normal and tumor samples, respectively. Investigating RNA-Seq data from the same seven TCGA liver samples, the HGT-ID workflow, identified eight HGT candidates in tumors and six HGT events in normal adjacent samples. Comparison of the HGT sites from WGS and RNA-Seq data has identified an overlap of six events in TCGA liver tumors .Using WGS tumor data, we identified Hepatitis B virus (HBV) integration events in six out of seven TCGA HCC tumors . No exogenous viral integration events were detected in these samples. Our results are consistent with the results reported in previous studies [The HGT-ID algorithm was applied to WGS data from 220 breast cancer samples collected by The Cancer Genome Atlas (TCGA) . As an example, for the HELA cell line sample, HGT-ID used only 4.3 h while VirusFinder2 with VERSE used 23.4 h. BATVI was not able to finish processing any of the four cervical cell line dataset in our system. Further, we compared the running time on the smaller simulation datasets for all three algorithms . HGT-ID demonstrated the fastest processing on the simulation datasets with highest coverage. The fast and accurate identification of HGT events by the HGT-ID workflow is primarily helpful in elucidating the effect of viral gene horizontal transfer on tumorigenesis and other diseases.We compared the computational performance of our workflow with VirusFinder2 . Using the HPV dataset as an example, HGT-ID used on average 14% of the time required by VirusFinder2 with VERSE when running on the same machine with default settings .We applied the HGT-ID workflow to publicly available large cancer cohorts, such as TCGA, to study HCC and breast cancer. We have shown the applicability of the tool in HCC samples where we have both WGS and RNA-Seq data sets available. We have surveyed the breast cancer data set using our workflow and did not find any evidence of HGTs. Among all of the events detected by HGT-ID in this report, we found about ~ 50% of events occured in highly repetitive regions masked by RepeatMasker , like mihttp://kalarikrlab.org/Software/HGT-ID.html). In addition, for the HELA cell line sample, HGT-ID used only 4.3 h while VirusFinder2 with VERSE used 23.4 h. To study other cancers or diseases with WGS or RNA-Seq data, the researchers can easily download the workflow and process the data through the HGT-ID to detect additional HGT candidates. The user manual and workflow are available to download. The fast and accurate identification of HGT events by the HGT-ID workflow is primarily helpful in elucidating the effect of viral gene horizontal transfer on tumorigenesis and other diseases.We compared the computational performance of our workflow with VirusFinder2 . VERSE intends to capture the consensus sequence to cover possible mutation in the virus by performing de-novo assembly. However, executing the VirusFinder2 with the VERSE algorithm is very time-consuming. Using the HPV dataset as an example, HGT-ID used on average only 14% of the time required by VirusFinder2 with VERSE when running on the same machine with default settings : Linux or VMProgramming language: PERL, JAVA, R and BASHOther requirements: noneLicense: Open Source (MIT license)Any restrictions to use by non-academics: none


A cochlear implant is a small electronic device that provides a sense of sound for the user, which can be used unilaterally or bilaterally. Although there is advocacy for the benefits of binaural hearing, the high cost of cochlear implant raises the question of whether its additional benefits over the use of an acoustic hearing aid in the contralateral ear outweigh its costs. This cost-effectiveness analysis aimed to separately assess the cost-effectiveness of simultaneous and sequential bilateral cochlear implantations compared to bimodal hearing  in children with severe-to-profound sensorineural hearing loss in both ears from the Singapore healthcare payer perspective. Incremental quality-adjusted life year (QALYs) gained and costs associated with bilateral cochlear implants over the lifetime horizon were estimated based on a four-state Markov model. The analysis results showed that, at the 2017 mean cost, compared to bimodal hearing, patients receiving bilateral cochlear implants experienced more QALYs but incurred higher costs, resulting in an incremental cost-effectiveness ratio (ICER) of USD$60,607 per QALY gained for simultaneous bilateral cochlear implantation, and USD$81,782 per QALY gained for sequential bilateral cochlear implantation. The cost-effectiveness of bilateral cochlear implants is most sensitive to utility gain associated with second cochlear implant, and cost of bilateral cochlear implants. ICERs increased when the utility gain from bilateral cochlear implants decreased; ICERs exceeded USD$120,000 per QALY gained when the utility gain was halved from 0.03 to 0.015 in both simultaneous and sequential bilateral cochlear implantations. The choice of incremental utility gain associated with the second cochlear implant is an area of considerable uncertainty. In Singapore, about 1.7 per 1,000 babies are diagnosed with severe-to-profound hearing loss or deafness at a median age of diagnosis of 2.7 months , providiThe cochlear implant can be used alone as unilateral cochlear implant, combined with an acoustic hearing aid in the contralateral ear to give the bimodal configuration for hearing , or bilaterally. The use of cochlear implant can benefit incidental learning . HoweverIn recent times, there has been emphasis on the benefits of binaural hearing, and many have advocated the use of a second cochlear implant in clinically appropriate candidates –15. CompBilateral cochlear implants potentially herald greater promise in binaural hearing. Bilateral cochlear implants can offer benefits over bimodal hearing in sound localisation and spatial unmasking , 17. It As a high cost technology , the addThe aim of this study is to evaluate bilateral cochlear implants’ cost-effectiveness compared to bimodal hearing in children with severe-to-profound sensorineural hearing loss in both ears, from the Singapore healthcare payer perspective. As bilateral cochlear implants can take place simultaneously in a single surgical procedure , or sequentially in two different surgical procedures , both types of bilateral cochlear implantations are included as interventions and evaluated separately against bimodal hearing as the comparator in this study.The incremental cost-effectiveness ratio (ICER) is calculated by taking the incremental cost of intervention compared to comparator, divided by the incremental effectiveness of intervention compared to comparator . This paThe eligible population for bilateral cochlear implants is children (<18 years old) with severe-to-profound sensorineural hearing loss in both ears. Children born without a cochlea or without auditory nerves, and those with neurological damage that prevent the processing of auditory information are not eligible for cochlear implants. This analysis uses bimodal hearing as the control group compared with: (1) simultaneous bilateral cochlear implantation: two initial cochlear implants received (one in each ear) at 1 year old; and separately (2) sequential bilateral cochlear implantation: one initial cochlear implant received at 1 year old and the second one in the contralateral ear two years later.A microsimulation model was developed to evaluate the cost-effectiveness of both simultaneous and sequential bilateral cochlear implantations compared to bimodal hearing as the control group in children born with severe-to-profound sensorineural hearing loss in both ears. A four-state Markov model was developed using TreeAge Pro 2016  in consultation with local experts to ensure face validity see . The fouThe model’s starting population comprises children who are eligible for bimodal hearing or bilateral cochlear implants. Eligible children enter the model at the health state “Use of 1st internal device” after the cochlear implantation. During the use of cochlear implant, children could develop clinical and device-related events, including internal device failure, major complications, wound revision due to major post-surgical complications, explantation and re-implantation, and all-cause mortality. They would either remain in the same health state, or transit to the health state “Use of 2nd internal device” if they experience an internal device failure or a major complication requiring explantation (followed by re-implantation). Similarly, should a second internal device failure or a major complication occur, they would transit to the health state “Use of 3rd internal device”.Our model used a lifetime horizon and annual cycle length . An annual discounting rate of 3% was applied to both costs and benefits. All analyses were conducted from the healthcare payer perspective. Monte Carlo simulation was used to run the analyses.Hearing devices can experience device failure. As a cochlear implant consists of internal and external devices, device failures can be further separated into internal device failure or external device failure. Similar to hearing aid, replacement of the external cochlear implant device requires only a consultation with the audiologist without the need for surgery. Replacement of the internal cochlear implant device requires surgical explantation of the failed internal cochlear implant device and re-implantation of a new one. Transitions between health states in the model are due to internal device replacement as a result of major complications or internal device failure, or all-cause mortality , 33. TheThe reliability of cochlear implant refers to the length of time for which implants work before they need replacement. We used empirical survival data from the manufacturers’ cochlear implant reliability reports to derive the probability of cochlear implant internal device failure –37. All The health outcome measure is the incremental QALYs gained associated with bilateral cochlear implants use over a lifetime horizon. In the absence of local data, we applied the same method, values, and assumptions as Bond et al. (2009), with thAs the clinical pathways for children receiving bilateral cochlear implants and bimodal hearing are the same for pre-surgery assessment for the second cochlear implant in both simultaneous and sequential bilateral cochlear implants, and post-surgery switch-on, mapping, and habilitation, only incremental resource use and costs relative to bimodal hearing were included in the analyses. This is similar to the approach by Martin et al. (2017) which compared bilateral cochlear implantation with unilateral cochlear implantation . Local cAll relevant unit charges were obtained from manufacturer and public healthcare institutions in 2017. They were assumed to remain constant over the simulated model period.The units of resource use were determined with input from local experts, and they are shown in Deterministic one-way sensitivity analyses were conducted over the range of predefined values of the point estimates for model parameters (i.e. ±10% or feasible range) separately for simultaneous and sequential bilateral cochlear implantation. Since all simulated cochlear implant users would receive a replacement internal cochlear implant device following internal device failure or a major complication requiring explantation (and re-implantation), these variables were not assumed to alter the cochlear implant users’ utility except after transiting to the death state. The impact of replacing an acoustic hearing aid or the external cochlear implant device on utility levels was taken to be minimal due to the transient nature of replacement. The parameters included in the one-way sensitivity analyses were utility gain from the second cochlear implant in both simultaneous and sequential bilateral cochlear implantations, acquisition cost of cochlear implant system, cost of hearing aid, cost of cochlear implantation in one ear and cost of re-implantation. Results were plotted as a Tornado diagram with the variables arranged in descending order according to the extent of the parameter’s impact on the ICER.A multivariate probabilistic sensitivity analysis was performed, using 10,000 second-order Monte Carlo simulation. We assigned gamma distributions to all cost variables and beta distribution to utility variables, and assumed that the standard deviation of each parameter was set to its mean. As Singapore does not have an explicit willingness-to-pay threshold to determine whether a health technology represents good value for money, cost-effectiveness acceptability curves were generated to present the probability of two types of bilateral cochlear implantations and bimodal hearing being cost-effective at varying willingness-to-pay thresholds.In the base case analysis, compared to the control group , the ICERs for simultaneous and sequential bilateral cochlear implantations are $60,607 and $81,782 per QALY gained respectively .We performed one-way sensitivity analyses to identify the variables that impacted the ICERs the most. Our results showed that when simultaneous and sequential bilateral cochlear implantations were independently compared to bimodal hearing, the ICERs were most sensitive to the utility gain from the second cochlear implant and the cost of initial bilateral cochlear implants. When the incremental utility gain for the second cochlear implant was halved from 0.03 to 0.015, the ICER of simultaneous bilateral cochlear implantation almost doubled to $121,143 per QALY gained. When the cost of the initial bilateral cochlear implantation increased by 10%, the ICER of simultaneous bilateral cochlear implantation was $67,883 per QALY gained; when the cost was reduced by 10%, the ICER of simultaneous bilateral cochlear implantation was $53,330 per QALY gained see . Other cFor sequential bilateral cochlear implantation, the ICER increased to $163,575 per QALY gained when the incremental utility gain for the second cochlear implant was halved from 0.03 to 0.015, and to $89,553 per QALY gained when the cost of initial bilateral cochlear implants increased by 10%. When the cost was reduced by 10%, the ICER of sequential bilateral cochlear implantation reduced to $74,011 per QALY gained see . SimilarThe average costs and QALYs over the 10,000 Monte Carlo simulations were essentially similar to the deterministic base case results. The mean probabilistic ICER was $60,995 and $82,111 per QALY gained for simultaneous and sequential bilateral cochlear implantations respectively when they were separately compared to bimodal hearing. The Monte Carlo simulation results are presented as cost-effectiveness acceptability curves in Results from our cost-effectiveness model were fairly consistent with published economic evaluations on bilateral cochlear implantation in populations comprising 80% bimodal hearing users—Bond et al. (2009)  and SummThe choice of incremental utility gain associated with the second cochlear implant, whether for simultaneous or sequential bilateral cochlear implantation, is an area of considerable uncertainty. Previous studies have demonstrated greater gains associated with unilateral cochlear implant than bilateral hearing aids in hearing, speech perception and speech production, enabling children with profound hearing loss or deafness to interpret sounds, understand other people, be better understood, and function more safely in their environment . The impA recently published systematic review concluded that there was insufficient evidence to demonstrate consistent benefits when comparing bimodal hearing with simultaneous or sequential bilateral cochlear implantation in children . AlthougResults from the probabilistic sensitivity analysis yielded cost-effectiveness acceptability curves that showed that sequential bilateral cochlear implantation was dominated throughout the willingness-to-pay from $0 to $100,000 per QALY gained. This suggests that a delay of merely two years during the critical age of learning could diminish the accrual of benefits in QALYs to an extent that renders sequential bilateral cochlear implantation not cost-effective throughout the willingness-to-pay range. Sequential bilateral cochlear implantation was dominated by simultaneous bilateral cochlear implantation, mainly due to its slower accrual of QALYs and higher post-surgery habilitation costs.There are several limitations in this study. The first limitation relates to uncertainties regarding the real incremental benefits of the second cochlear implant in both simultaneous and sequential bilateral cochlear implantations compared with bimodal hearing. In light of the considerable uncertainty surrounding the choice of incremental utility gain, and the lack of supporting evidence, we were unable to incorporate the effects of education and career achievement, whether in the form of changes in utility gain or productivity loss. More longitudinal research in paediatric patients comparing bimodal hearing with bilateral cochlear implants in utility changes and clinically-relevant long-term outcomes that track functional performance, psychosocial well-being, and achievements in academics and career will help to mitigate the uncertainty.Second, we are aware that a constant utility gain over a long time horizon may conceal possible changes in utility associated with hearing loss or deafness as a result of severity of condition and psychosocial adjustment over time. This may potentially overestimate the benefits of the second cochlear implant in both simultaneous and sequential bilateral cochlear implantations for children.Third, as the utility scores reported in existing literature were generated in western countries, it is unclear if they could be readily generalised to the Singapore context, particularly if they have communities with a stronger Deaf culture, or different perception of benefits across cultural groups, such as the child’s academic progression, self-esteem, and stress levels.Fourth, device failure rates were derived from survival data of all internal devices found in manufacturers’ online reliability reports, with follow-up periods ranging from 2 to 19 years. The earlier the device was launched, the longer their survival data, which implied that a greater weight was conferred to older models in the estimation of reliability. Yet, with continuous advancements in cochlear implant technologies, the more recently launched cochlear implants are expected to have a lower failure rate. This could potentially overestimate the average failure rate of the internal device. However, since only two internal cochlear implant device replacements were allowed over the simulated lifetime in the model, the impact of this overestimation on the ICERs would be negligible.Fifth, we were unable to account for the effects of any incremental innovation associated with newer hearing aid technologies. Hearing aids vary in the number of sound processing channels and how well they can filter background noises to enhance speech signals. When used in the bimodal way, advanced hearing aids are equipped with features to share sound signals received by both devices to enhance the binaural experience , 46. TheIn children with severe-to-profound sensorineural hearing loss, compared to bimodal hearing, simultaneous bilateral cochlear implantation gave an ICER of $60,607 per QALY gained, while sequential bilateral cochlear implantation gave an ICER of $81,782 per QALY gained. Sensitivity analyses found that the results were most sensitive to utility gain associated with the second cochlear implant, and cost of bilateral cochlear implants. The choice of incremental utility gain associated with the second cochlear implant, whether for simultaneous or sequential bilateral cochlear implantation, is an area of considerable uncertainty. In view of the considerable uncertainty in the incremental clinical benefits, bilateral cochlear implantation may not represent good value for limited healthcare dollars in Singapore context unless the cost of bilateral cochlear implants is appropriately reduced.S1 File(DOCX)Click here for additional data file.


The World Health Organization (WHO) estimates that 466 million people experience moderate or higher levels of hearing loss and that this number could double by 2050 if the current demographic trends continue.Prevention of deafness and hearing lossFortunately, many of the causes that lead to hearing loss can be avoided through preventative public health actions. Those who experience hearing loss can benefit from early identification and intervention. Hearing aids, cochlear implants, use of sign language and assistive technologies are some of the available options to ensure that no one lives with unaddressed hearing loss. The 2017 World Health Assembly Resolution ,These challenges can be tackled through the integration of ear and hearing care into health systems as part of universal health coverage (UHC). In 2015, when the United Nations General Assembly adopted the sustainable development goals, WHO and its Member States committed to achieve UHC by 2030.–,,,,This theme issue draws further attention towards this topic, highlighting the impact of unaddressed hearing lossWorld report on hearing, which will recommend a package of evidence-based interventions for improving ear and hearing care through health system strengthening.The theme issue sets the stage for the First Membership Assembly of the World Hearing Forum, which will take place this December at WHO headquarters in Geneva, Switzerland.


To study the correlation of hearing loss with depression, anxiety and stress in patients suffering from chronic suppurative otitis media in local population of Pakistan.This is a cross-sectional study conducted from May to September 2018 at tertiary care hospital of Karachi. One hundred and twenty patients of chronic suppurative otitis media were divided into three groups: Group-1 (maintained on ciprofloxacin), Group-2 (maintained on co-amoxicillin) and Group-3 (did not subject to the treatment).The measurement of hearing loss was carried out by pure tone audiometry (PTA) and the depression, anxiety and stress were scored taking depression, anxiety and stress scale (DASS) as tool. To observe the effect of hearing loss on different groups one way ANOVA was applied and Spearman correlation was used to find correlation of depression with hearing loss.There was no significant difference found for hearing loss and severity among the groups treated with ciprofloxacin, co-amoxicillin and not maintained on antibiotic therapy. Positive correlations found between hearing loss and depression, anxiety and stress in patients of the three groups.Depression induced by hearing loss as a result of CSOM in patients need to be monitored during and after treatment and scored so that can be treated by counseling and antidepressant (if required). Information regarding this topic on population of Pakistan will be helpful for health care takers and policy makers to manage mental stress with hearing loss in CSOM. Chronic suppurative otitis media (CSOM) is a middle ear inflammation usually accompanied with infection, leads to perforation of tympanic membrane and prevalent up to 72 cases in 1000 inhabitant in developing countries. According to estimation over two billion dollars are being consumed to treat ear infection in United States of America.3Many studies have focused the association of depression with hearing loss and CSOM one of the leading factors of decreased hearing capability across the world. Risk factors such as socio-economical, duration of disease and occupational stress influence the characteristic audiologic alteration in the patients of CSOM and also contribute in depression.6A very interesting study conducted on tinnitus patients with hearing loss to evaluate the co-morbidities of depression, stress and anxiety, taking depression, anxiety and stress scale (DASS) as tool to determine the correlation of depression, stress and anxiety with hearing loss in tinnitus patients. Although different scales are available to measure depression but psychometric properties of DASS showed better separation in factor loadings as compare to other scales and its tripartite model of depression, anxiety and stress make it possible to differentiate these conditions.7Unfortunately very few studies have been conducted on CSOM patients to focus the co-relation or association of depression with hearing loss all over the world but in local population of Pakistan no authentic data is available which satisfy the sample size of the study, therefore, our objective was to evaluate the correlation of hearing loss with depression, anxiety and stress in patients suffering from chronic suppurative otitis media in local population of Pakistan.The present study was reviewed and approved by Institutional Review Board of Jinnah Sindh Medical University and conducted in ear, nose and throat department of a tertiary health care in accordance with the relevant regulations. This is a cross-sectional research on patients with the clinical presentation of CSOM subjected to PTA after evaluation of medical history and examination with otoscope.patients aged between 18-75 years, both the genders having unilateral ear presentation of CSOM without fluid discharge at the time of pure tone audiometry.paediatric population and patients above 75 years with the history of neurological disorder or profound psychological distress, cardiac arrest, family history of sensoneural hearing loss or using hearing aid.Sample size calculation; the minimum sample size was calculated (n=68) with the help of OPEN-EPI, keeping 6.3% prevalence of CSOM and 95% confidence interval, taking 5% margin of error PTA was examined at differences frequencies using an audiometer with aural headphones to measure hearing thresholds and divide into seven categories as normal (25 db), mild (26 to 34 db) HL, moderate (50-64 db) HL, severe 65-79 db) HL, profound (80 to 94 db) HL and deaf.9 db HL, Associations between hearing loss and depression was evaluated by Spearman correlation test. The effect of hearing loss (HL) on different groups was compared (on both the genders) using one way ANOVA of independence (p< 0.01). In order to check the co-relation among different variables such as age, gender, hearing loss type (HLT) and hearing loss severity (HLS) Pearson correlation was applied.One hundred twenty patients of CSOM of both genders were equally divided into three groups in the current study as shown in No significant difference was observed for hearing loss severity and hearing loss type for groups as shown in The results of PTA with the corresponding groups are presented in mean±S.D in The positive correlation of depression, anxiety and stress with both the genders in CSOM patient was observed as shown in In this study we had selected 120 patients of both gender in ratio of 1:1 of CSOM and divided them into three groups (40 patients per group) keeping prevalence rate of South AsiaGender of the patients was significantly correlated with the hearing loss type (p=0.001) in our data and 9In our study significant correlation of age was observed with hearing loss severity (p=0.002) in our study and 10Patients of Group-1 (maintained on ciprofloxacin) had average hearing threshold of 43.88 db, Group-2 (treated with co-amoxicillin) had 48.72 db and Group-3 (without any treatment) showed 50.44 db, poorer than normal threshold levels as reported in other studies conducted on clinical samples.Although an earlier study showed that 13% cases of Sensorineural Hearing Loss found in CSOM1216Spearman’s correlation proved the positive correlation among different variables taken under consideration including hearing loss type (hlt), age and hearing loss severity (hls) with depression, anxiety and stress. In addition to this, female seemed to developed symptoms of depression and anxiety more than male as shown in 17The significant depression found in all three groups  showed the behavioral changes of the patients of CSOM due to the frustration with the condition of hopelessness, devaluation of life and lack of motivation. It was also observed in another observational study conducted to evaluate the association of hearing loss with cognition and depression on 5043 patients that hearing loss increased the risk of depression.4Therefore, we are agree with Hasson et al.Data on depression, anxiety and stress was based on self-reported questionnaires which is limited to the past week experience based responses and majority of the respondents had low literacy profile. Patients with the family history of hearing loss or depression were not included.Depression induced by hearing loss as a result of CSOM in patients should be scored during and after treatment and risk factors should be focused so that can be treated by counseling and antidepressant (if required). It will guide the care takers to cure the patient mentally and physically. This study will help the health providers and researchers for the better management of the consequences of CSOM.SMK conceived, data collection, designed and manuscript writing.SMTR did review and final approval of manuscript.NA did data collection.M editing of manuscript.


Hearing disorders are usually unilateral and are more common in women. They canbe congenital or acquired, and hearing loss is categorized as sensorineural,conductive, or mixed. The onset of hearing loss can be progressive or sudden,and it is a common reason for seeking medical attention. In this context,computed tomography and magnetic resonance imaging have assumed critical rolesin the search for an etiological diagnosis and in guiding the therapeuticapproach. In this pictorial essay, we illustrate the common causes of hearingloss, discussing the possible differential diagnoses and highlighting the mostrelevant imaging findings. Hearing is of fundamental importance in the overall development of human beings, asit is considered one of the main senses. Any disorder that affects it, to anydegree, has an adverse effect on functional status, quality of life, and cognitivefunction, as well as on emotional, behavioral, and social well-being.During the diagnostic investigation, computed tomography (CT) and magnetic resonanceimaging (MRI) of the temporal bone and of the brain play a fundamental role in alarge portion of hearing disorders, helping to define the therapeutic approach.There are many etiologies associated with hearing loss. In the initial evaluation,psychiatric and pharmacological causes should be excluded before any imagingevaluation is considered.In this study, we will discuss the imaging findings according to the etiologicalclasses, organized by cause: neoplastic, infectious/inflammatory, congenital,traumatic/post-surgical, and other.A schwannoma is the most common tumor of the cerebellopontine angle, usuallypresenting a homogeneous aspect when small. Its insinuation into and enlargementof the internal auditory canal is a suggestive but nonspecific aspect . The affParagangliomas are tumors of the chemoreceptor system and are the main primarytumors of the jugular foramen. Although most paragangliomas are benign, theybehave aggressively. On CT, they manifest as irregular bone destruction andsignificant contrast enhancement . On MRI,(1). On CT, the bone destructionis either geographic or in a moth-eaten pattern, with a halo of peripheralcalcification. On MRI, the signal is heterogeneous, with hyperintense fociwithin the lesion seen in T1-weighted sequences , with or without labyrinthine fistulae . A functOtomastoiditis and osteomyelitis consist of infection of the tympanic and mastoidcavities, mainly caused by bacteria. Immunocompromised patients present withrisk factors for unusual infectious agents, in addition to more extensive andrapidly progressive impairment. On CT, uncomplicated otomastoiditis commonlymanifests as hypodense material with no bone erosion. On MRI, no restricteddiffusion is expected. When not properly treated, otomastoiditis can evolve toosteomyelitis or intracranial complications, including meningitis, abscesses,and venous thrombosis . Theinc. MRIfindings precede those of CT by some months, and are characterized by loss ofthe fluid signal within the membranous labyrinth ,trauma, tumors, hemorrhage, otosclerosis, sickle cell anemia, or otherfactors. MRIfinabyrinth  and gadoabyrinth ..An enlarged vestibular aqueduct is the most common congenital abnormality of thetemporal bone and is associated with sensorineural hearing loss. It may beunilateral or bilateral, typically being identified at birth or in the firstyears of life. It occurs in isolation or in conjunction with syndromes. On CT,it is characterized by enlargement of the bone canal that extends from thevestibule to the posterior surface of the petrous portion of the temporal bone, vestibu,4..Cochlear malformations are often accompanied by vestibular alterations .Most temporal bone fractures result from high-energy traumatic brain injury. Thetraditional classification indicates a relationship between the fracture lineand the longest axis of the petrous portion of the temporal bone, thesefractures thus being categorized as longitudinal, transverse, or mixed.Longitudinal fractures usually occur in temporoparietal trauma, affecting mainlythe extralabyrinthine portion, the main complications being ossicular lesion andhemotympanum . Transve,8., as depicted in Stapedectomy and reconstruction of the stapes is often used in order to restoreconductive hearing loss in patients with otosclerosis or congenital anomalies.An ossicular prosthesis can malfunction weeks to years after surgery. CT helpsidentify the most common repairable causes of prosthesis failure, such assubluxation or luxation, prosthesis migration , and displacement of the incudomalleolar joint, as depi(9). In the fenestral type, noncontrast CT is the modalityof choice and shows involvement of the lateral wall of the inner ear, mainlyalong the oval and round windows, the fissula ante fenestram being the regionthat is most affected . There are two types ofotosclerosis-fenestral and retrofenestral/cochlear-the first being more relatedto conductive hearing loss and the second being related to sensorineural ormixed hearing lossaffected . In the. Deafness is the most common sensory deficit,occurring in 30-50% of patients. On CT, Paget's disease manifests as corticalthickening and coarse trabeculation, with bone expansion and irregularities(.Paget's disease is a focal disorder of bone remodeling, resulting in adisorganized, thickened bone structure with reduced mineral density. It isrelatively common in the elderly population, and there is involvement of thetemporal bone in 65-70% of the cases. Deafnes patients. On CT, larities. On T1-warities(.CT and MRI are useful tools in the evaluation of patients with hearing loss and mayadd information essential to the diagnosis, treatment planning, and follow-up. Theradiologist must be attentive to their indications, in order to be able tocontribute to the clinical decision-making process.