The change in rhizosphere soil bacterial community and root system under new water-saving device is not clear.A completely randomized experimental design was used to explore the effects of different micropore group spacing  and capillary arrangement density  on tomato rhizosphere soil bacteria community, roots and tomato yield under MSPF. The bacteria in tomato rhizosphere soil were sequenced by 16S rRNA gene amplicon metagenomic sequencing technology, the interaction of bacterial community, root system and yield in tomato rhizosphere soil was quantitatively described based on regression analysis.Results showed that L1 was not only beneficial to the development of tomato root morphology, but also promoted the ACE index of tomato soil bacterial community structure and the abundance of nitrogen and phosphorus metabolism functional genes. The yield and crop water use efficiency (WUE) of spring tomato and autumn tomato in L1 were about 14.15% and 11.27%, 12.64% and 10.35% higher than those in L2. With the decrease of capillary arrangement density, the diversity of bacterial community structure in tomato rhizosphere soil decreased, and the abundance of nitrogen and phosphorus metabolism functional genes of soil bacteria also decreased. The small abundance of soil bacterial functional genes limited the absorption of soil nutrients by tomato roots and roots morphological development. The yield and crop water use efficiency of spring and autumn tomato in C2 were significantly higher than those in C3 about 34.76% and 15.23%, 31.94% and 13.91%, respectively. The positive interaction between soil bacterial community and root morphological development of tomato was promoted by the capillary layout measures of MSPF.The L1C2 treatment had a stable bacterial community structure and good root morphological development, which positively promoted the increase of tomato yield. The interaction between soil microorganisms and roots of tomato was regulated by optimizing the layout measures of MSPF to provide data support for water-saving and yield-increasing of tomato in Northwest China. Soil baFacility Planting agriculture has the characteristics of fast crop growth and high demand for soil water and fertilizer . MeasuriRedistribution of soil moisture, heat and air is closely related to field irrigation management, which directly or indirectly affects the development of soil microbial community and plant root morphological development . In ordeAt present, the mechanism of the change of crop soil bacterial community and root system regulated by the new water-saving technology of facility planting agriculture under MSPF  is not c22.1The experiment was carried out in the greenhouse of Xi ‘an Modern Agricultural Science and Technology Exhibition Center  in Shaanxi Province from March 23, 2019 to January 30, 2020. The tested tomato variety was ‘Jingfan 401’ . The tomato adopts the ridge planting structure mode, in which the row spacing is 50.00 cm and the plant spacing is 40.00 cm. The length of the experimental plot is 3.40 m, and the spacing between the plots is 4.00 m. The planting, irrigation and harvest time of spring tomato and autumn tomato are shown in In this experiment, the irrigation amount was controlled on the basis of the cumulative evaporation from a 20-cm diameter standard pan  following Zhang . The evapanE represents the evaporation within the interval of two irrigation, based on the cumulative evaporation from a 20 cm diameter pan (mm); A represents the capillary control area (mm) and kcp represents the crop- pan coefficient. In this paper, adopting adequate irrigation mode, the crop-pan coefficient of is 1.0  Soil samples were collected from rhizosphere soil of spring tomato and autumn tomato after 72 days of planting. The soil shaking method was used to extract . Three rhizosphere soil samples were randomly taken from each experimental plot, and the samples were transported to the laboratory on ice. The samples were brought back to the room and the fresh soil plant residues were removed. Three soil samples in the plot were fully stirred and evenly mixed as soil samples for sequencing in the experimental plot. The soil samples were collected and quickly frozen in liquid nitrogen. The soil samples were stored in a -80°C refrigerator and sent to Shanghai Meiji Biomedical Technology Co., Ltd.  to determine soil bacterial community. The main analysis steps of soil bacterial community determination are divided into three parts:A. DNA extraction and PCR amplification® soil kit , which reliably and quickly separates high-quality genomic DNA from various soil samples (up to 1 g of soil can be processed in 60 min). After the quality of DNA extraction was detected by 1% agarose gel electrophoresis, the V3-V4 variable region was amplified by PCR with 338 F (5 ‘ -ACTCCTACGGGAGGGAGCAGCAG-3 ‘) and 806 R (5 ‘ -GGACTACHVGGGTWTCTAAT-3 ‘) primers. The amplification procedure was: 95 °C pre-denaturation 3 min, 27 cycles . Finally, extension at 72 °C for 10 min (PCR instrument: ABI GeneAmp ® 9700).Total DNA was extracted from tomato rhizosphere soil using the E.Z.N.A.Â B. Illumina Miseq sequencing2O was added to make up to 25 μL. Reaction parameters: 95 °C for 5 min; 95 °C denaturation 45 s, 55 °C annealing 50 s, 72 °C extension 45 s, 28 cycles; 72 °C for 10 min. The original sequence was uploaded to the figshare database .The bacterial 16S rDNA V3-V4 region was selected, and the NA samples were sequenced using the Illumina Miseq PE300 high-throughput sequencing platform . The bacterial 16 S rDNA V3-V4 amplification primers were 338 F (5 ‘ -ACTCCTACGGGGAGGCAGCAG-3 ‘) and 806 R (5 ‘ -GGACTACNNGGGTATCTAAT-3 ‘). The PCR products were recovered using 2% agarose gel, purified and eluted for detection. PCR reaction system : 12.5 μL KAPA 2G Robust Hot Start Ready Mix, 1 μL Forward Primer (5 μmol/L), 1 μL Reverse Primer (5 μmol/L), 5 μL DNA , and finally 5.5 μL dd HC. Sequencing data processinghttps://login.majorbio.com/login).The original data obtained by Miseq sequencing were optimized after splicing and quality control. After distinguishing the samples, OTU  cluster analysis and species taxonomy analysis were performed. The OUT similarity was set to 97%. The OTU was subjected to diversity index analysis and statistical analysis of community structure at each classification level, and then a series of in-depth statistical and visual analysis such as multivariate analysis and difference significance test of sample community composition and phylogenetic information were completed. The ACE index of soil bacterial (ACE), CHAO index of soil bacterial (CHAO), COVERAGE index of soil bacterial (COVERAGE), SHANNON index of soil bacterial (SHANNON), SIMPSON index of soil bacterial (SIMPSON), SOBS index of soil bacterial (SOBS), Species Veen diagram analysis and species composition analysis can be obtained by direct analysis of Shanghai Meiji Biological Cloud platform (2) Based on the results of soil bacterial community determination, and referring to the Kyoto Encyclopedia of Genes and Genomes and related references , the fun2.3.2Three tomato plants were randomly selected from each plot to dig a soil volume with a depth of about 0.4 m and a diameter of 0.2 m centered on the plant at 76 and 78 days after planting spring tomato and autumn tomato, respectively. The samples were placed in a 150 mesh sieve to rinse the roots. The roots were scanned by Epson Perfection V700 scanner to obtain the TIF diagram. Finally, the TIF diagram was processed by WinRHIZO Pro software to obtain the total root length, total number of root tips and bifurcation number of greenhouse tomatoes. The root activity of tomato was determined by triphenyltetrazolium chloride method .2.3.3Four tomato plants were randomly selected, and the mature fruit mass of four tomatoes was weighed by electronic scale with precision of 0.01 g, and the yield per hectare was converted.ETa )and crop water use efficiency (WUE) were calculated formulas (2) and (3), respectively  was used to measure the soil volume moisture content at different layers of soil . It was measured once before and after each growth period. Water consumption (ectively :I represents the irrigation quota of crop growth period (mm); H represents the depth of the wetting layer with plan (H = 0.8 m); θt1 and θt2 represent 80-cm average soil volumetric water contents at times  t1 and t2 cm3/cm3), respectively.represents crop water consumption during growth period (mm); Y indicates crop grain yield (t/hm2).2.4P<0.05. The picture was drawn by OriginPro2019 . Excel 2016  was used for regression analysis.The interaction of bacterial community, root system and yield in tomato rhizosphere soil was quantitatively described based on regression analysis. The significant difference was analyzed by F test of SPSS22.0 , and the significant level was set to 2.5The abbreviations of this article are explained in 33.13.1.1It can be seen from the dilution curve of Compared with L2, the ACE, CHAO, SHANNON, and SOBS of spring tomato and autumn tomato treated with L1 were higher. With the decrease of C, the ACE, CHAO, SHANNON, SIMPSON and SOBS of soil bacterial with spring tomato and autumn tomato showed a decreasing trend. The diversity of soil bacterial community structure in spring tomato was lower than that in autumn tomato.3.1.2As can be seen from Bacillus (10.71%-23.68%), Streptomyces (1.44%-3.17%), Sphingomonas (1.40%-3.21%); the soil of autumn tomato mainly includes Sphingomonas (6.07%-10.68%), Bacillus (2.55%-6.85%) and Gemmatimonas (1.75%-2.65%). Bacillus is a common genus of spring tomato and autumn tomato soil. With the decrease of C, the abundance of Bacillus in spring and autumn tomato increased first and then decreased. Compared with L2, the abundance of Bacillus with spring and autumnin L1 treatment increased by 23.50% and 76.17% at genus level.From 3.2From Compared with L2, the NFFA, NFA, soil bacterial denitrification functional gene abundance (DFA), GAN and GAP in spring tomato and autumn tomato soil treated with L1 increased by 44.14% and 10.44%, 5.85% and 16.27%, -0.85% and 4.64%, 11.00% and 8.99%, 6.69% and 15.34%, respectively. With the decrease of C, the NFFA, NFA, DFA, GAN and GAP in spring tomato and autumn tomato soil increased first and then decreased. The NFFA, NFA, DFA, GAN and GAP in C2 of spring tomato and autumn tomato soil was significantly higher than that in C3 about 31.21% and 40.78%, 8.11% and 24.10%, 10.59% and 15.74%, 14.83% and 26.36%, 27.97% and 19.29%, respectively. The abundance of soil bacterial nitrogen metabolism and phosphorus metabolism functional genes in spring tomato was lower than that in autumn tomato.3.3P ≤ 0.05). The relative contribution of C to RL, RS, RV, RT, RB and RA of tomato was 16.10%, 16.40%, 16.10%, 18.60%, 17.50% and 13.20%, respectively. The D had a significant effect on RL, RS, RV, RT, RB and RA of spring tomato and autumn tomato (P ≤ 0.05). The relative contribution of D to RL, RS, RV, RT, RB and RA of tomato was 23.00%, 22.60%, 13.20%, 10.30%, 9.70% and 13.20%, respectively.It can be seen from Compared with L2, the RL, RS, RV, RT, RB and RA of spring tomato and autumn tomato in L1 increased by 2.62% and 4.19%, 2.87% and 7.05%, 3.45% and 8.49%, 3.83% and 2.98%, 10.87% and 8.25%, 8.31% and 5.94%, respectively. With the decrease of C, the RL, RS, RV, RT, RB and RA of spring tomato and autumn tomato increased first and then decreased. The RL, RS, RV, RT, RB and RA of spring tomato and autumn tomato in C2 treatment were about 1.06% and 1.76%, 1.22% and 6.28%, 1.76% and 14.07%, 0.14% and 4.39%, 2.77% and 0.18%, 2.67% and 1.04% higher than those in C1 treatment. It was also higher than C3 treatment by about 12.37% and 12.62%, 22.21% and 20.25%, 41.73% and 45.63%, 20.05% and 16.68%, 46.82% and 21.05%, 20.42% and 20.23%, respectively. The root morphological development of spring tomato was better than that of autumn tomato.3.4It can be seen from 3.5Based on Pearson’s two-tailed test, the indexes with better correlation among soil bacterial community, root system, and yield were screened out. Correlation analysis Figure 92>0.6015, indicating that the ACE in the regression model can explain 60.15% of the change of tomato RT. The GAN and GAP also showed a quadratic parabolic curve relationship with the RT of tomato. The determination coefficient R2 of the regression equation between the GAN and the RT of tomato was higher than that of the determination coefficient R2 of the regression equation between the GAP and the RT of tomato, indicating that the GAN had a greater impact on the RT of tomato. The relationship between the RT of tomato and the yield was a quadratic parabolic curve, and the coefficient of determination R2>0.6461, indicating that the RT of tomato in the regression model could explain 64.61% of the change in yield of tomato. The RT of tomato could be used to estimate the yield, and the tomato production potential in this area could be indirectly evaluated by soil bacterial community.From 44.1Previous studies have shown that under drought stress, plants will reduce the supply of carbon sources for soil bacteria, and sustainably reduce the diversity and richness of bacterial community structure . It was The C can change the composition of microbial community by changing the wet/dry conditions of soil, thus changing the mineralization rate of soil nutrients and affecting the absorption capacity of crop roots to nutrients . This st4.2The effect of C change of MSPF on soil wetted body is similar to that of dripper spacing change . There iThis study found that too high or too low capillary density was not conducive to tomato root morphological development. It may be due to the increase of the outflow cross section per unit area in the test area when the C is too high (C1), which is easy to be saturated in shallow soil, form water accumulation layer on the surface, restrict the exchange of soil air and external gas, produce hypoxia stress and restrict root growth . When th4.3Previous studies have found that the difference in soil water distribution caused by irrigation can significantly affect soil bacterial community, crop root morphological development, and increase or decrease soil carbon, nitrogen and phosphorus metabolic activity . It was Previous studies have found that there is an interactive relationship between tomato root morphological development and soil microorganisms . In this5By exploring the layout measures of MSPF to regulate the soil bacterial community and root morphological development of tomato in greenhouse, it was found that the diversity and abundance of soil bacterial community structure of spring tomato and autumn tomato in L1 were higher than those of L2. Among them, the total number of OTUs classification of soil bacteria in spring tomato and autumn tomato with L1 was higher than that of L2 by about 13.50% and 6.16%, respectively, and the ACE in spring tomato and autumn tomato with L1 was higher than that of L2 about 6.90% and 8.19%, respectively. The GAN and GAP in spring tomato and autumn tomato soil with L1 was higher than that of L2 about 11.00% and 8.99%, 6.69% and 15.34%, respectively. As a result, the yield and water use efficiency of spring tomato and autumn tomato treated with L1 were higher than those of L2 by about 14.15% and 11.27%, 12.64% and 10.35%, respectively. With the decrease of C, the diversity of soil bacterial community structure decreased significantly. Among them, the ACE and total number of OTUs classification of soil bacteria with C2 was significantly higher than that of C3 about 27.59% and 9.85%, 30.68% and 16.21%, respectively. The GAN and GAP decreased with the decrease of C. For example, the GAN and GAP in soil of spring tomato and autumn tomato with C2 was significantly higher than that of C3 about 14.83% and 26.36%, 27.97% and 19.29%, respectively. Lower soil bacterial community structure diversity and soil bacterial nitrogen and phosphorus metabolism functional gene abundance reduced greenhouse tomato soil nitrogen and phosphorus cycle. As a result, the yield and water use efficiency of spring and autumn tomatoes with C2 were significantly higher than those of C3 by 34.76% and 15.23%, 31.94% and 13.91%, respectively. Pearson two-tailed test and regression analysis showed that there was a positive interaction between soil bacterial community and root morphological development. The relationship between soil bacterial community and RT was a quadratic curve, and the relationship between RT and yield was also quadratic curve, indicating that the RT of tomato could be used to estimate the yield, and the tomato production potential in this area could be indirectly evaluated by soil bacterial community. This study provides a reference for regulating tomato root system and soil microbial interaction and increasing tomato yield by optimizing the layout measures of MSPF.https://figshare.com/articles/dataset/The_Layout_Measures_of_Micro-sprinkler_Irrigation_under_Plastic_Film_Regulate_Tomato_Soil_Bacterial_Community_and_Root_System/21818610.The data presented in the study are deposited in the figshare repository, MZ wrote and revised the manuscript. YL participated in the experiments. NX, HY, FG, and JL collected the materials and analyzed the data. YL, ZZ and MZ conceived and designed the research. All authors contributed to the article and approved the submitted version.


Dryococelus australis, a critically endangered Australian phasmid. The assembly, constructed with Pacific Biosciences continuous long reads and chromatin conformation capture (Omni-C) data, is 3.42 Gb in length with a scaffold N50 of 262.27 Mb and L50 of 5. Over 99% of the assembly is contained in 17 major scaffolds, which corresponds to the species’ karyotype. The assembly contains 96.3% of insect Benchmarking Unique Single Copy Ortholog genes in single copy. A custom repeat library identified 63.29% of the genome covered by repetitive elements; most were not identifiable based on similarity to sequences in existing databases. A total of 33,793 putative protein-coding genes were annotated. Despite the high contiguity and single-copy Benchmarking Unique Single Copy Ortholog content of the assembly, over 1 Gb of the flow-cytometry-estimated genome size is not represented, likely due to the large and repetitive nature of the genome. We identified the X chromosome with a coverage-based analysis and searched for homologs of genes known to be X-linked across the genus Timema. We found 59% of these genes on the putative X chromosome, indicating strong conservation of X-chromosomal content across 120 million years of phasmid evolution.We present a chromosome-scale genome assembly for  Timema, is the outgroup of all other extant stick insects, and so the diversity of the order is severely underrepresented. Dryococelus australis is a critically endangered Australian stick insect, currently bred in captivity and with a highly restricted range and population size in the wild. Population genetic studies of the species have been recommended for over a decade now. To this end, we present a chromosome-scale genome assembly for D. australis. This assembly is highly complete and assembled into scaffolds corresponding to the species’ karyotype. This assembly significantly expands the genomic resources available for the study of phasmids and will facilitate future population genomic studies of an iconic Australian insect and flagship species for invertebrate conservation.Stick insects are a diverse insect order comprising over 3,000 recorded species, yet only one genus is represented among publicly available chromosome-scale assemblies. This genus, Dryococelus australis, the Lord Howe Island (LHI) stick insect, is a critically endangered  in 1918. In 2001, a population was discovered on Ball's Pyramid (BP), a sea stack about 20 km southeast of LHI (GCA_002236955.1), which showed signs of polyploidy but also had very low contiguity . In this study, we present a chromosome-scale, annotated genome assembly for D. australis and place the species in a phylogenetic context. We also investigate X chromosome conservation by leveraging existing phasmid genomic data. Presently, the only published chromosome-scale phasmid assembly is of Timema cristinae (GCA_002928295.1), a genus that forms the outgroup to all other phasmids and which is estimated to have diverged from D. australis 121.8  million years ago . C-banding demonstrated mostly uninformative heterochromatin distributions with only the centromeres differentiated except for two chromosomes per individual that differentially stained across their entire length (b). We infer these to be the sex chromosomes. The sex of the embryos dissected was unknown but the individual depicted in figure 1b has one large and one small putative sex chromosome, implying XY sex determination which has been observed in other stick insects (Direct observation of chromosomes identified a diploid karyotype of 2N = 34 a. C-bande length b. We inf insects .a and b). We mapped the OmniC reads back to these scaffolds and plotted the resultant contact matrix (fig. 1b). We assessed completeness of the scaffold assembly by searching for Benchmarking Unique Single Copy Ortholog (BUSCO) genes from the insecta_odb10 database. The genome contains complete single-copy sequences of 96.3% of insect BUSCOs with 0.7% present but duplicated  with WTDBG2 followed by removal of potential contaminants and secondary haplotypes generated an assembly of 2,788 contigs with N50 and L50 values of 13.48 Mb and 76 . Mappingfig. 1t matrix  online. plicated . Only 46plicated .Clitarchus hookeri, n = 66,470 [Medauroidea extradentata, n = 35,742 [Timema annotations tend to have 11,000 to 16,000  of the assembly was masked by RepeatMasker ; howeveCO genes  althoughediction . As a reatMasker  online. atMasker  online. atMasker  online, atMasker  online. atMasker  online.D. australis population (from which the assembly presented herein was derived) has experienced a recent colonization bottleneck and subsequent low population size  transposition  of the true genome sequence remains unassembled. Recent bursts of transposable and repetitive elements, which we believe is likely and is borne out by the divergence landscapes of the major repeat classes . This places D. australis within the Australian Lanceocercata, corroborating previous findings based on mitochondrial genomes ;  missing  online. ustralis c. This p genomes  and Sang genomes , 2010 thTimema species (d), and the remainder mapped to the other major scaffolds mostly in proportion to their length, for example, the next scaffold with the most hits was Scaffold_1, the longest scaffold. Scaffold_11 is a notable outlier to this trend (d), harboring 10 best hits compared with Scaffold_3 which harbored 9, despite being almost three times as long. We also remapped the PacBio reads generated from the male sample used in the initial assembly back to the major scaffolds and calculated GC normalized depth in 10 kb bins. This same scaffold had roughly 50% mean depth of coverage compared with the other scaffolds (e). Finally, Scaffold_4 is also an outlier in repetitive element composition, overall and for specific element types  suggests the presence of a Y chromosome not apparent in the assembly. We attempted polymerase chain reaction (PCR) primer design from sequences of minor scaffolds showing inflated coverage in male versus female samples (with sequencing reads from another ongoing project) but no primers showed successful differential amplification from male and female gDNA extracts, and so we are unable to confirm the true sex determination system of D. australis.We used tblastn to identify putative homologs of X-linked genes which are conserved across five  species . The maj species d, and this trend d, harborcaffolds e. Finallnt types  online.  embryos b suggestD. australis. Eggs from the captive colony were dissected at 2–3 months after laying and the embryos placed in distilled water for 10 min, washed twice in a 3:1 solution of methanol and glacial acetic acid, and finally incubated in this same solution for 2 h at room temperature. Embryos were then macerated on a glass microscope slide in a 3:2 solution of glacial acetic acid and distilled water using a blunt brass rod. Extraneous material was removed with forceps and the cellular suspension rolled across the surface of the slide. The slide was dried on a hotplate set to 35 °C for 15 min. Slides were then incubated in saturated BaOH solution for 5 min followed by washes in distilled water, 0.2 M HCl, distilled water again, and a final 30 min incubation in 2X saline sodium citrate at room temperature. Slides were washed with a solution of 1% w/v KH2PO4 2% w/v Na2HPO412H2O and stained in a 5% v/v solution of Giemsa in the same solution. Slides were visualized at 1,000× with a compound light microscope and imaged with a mounted Nikon D90.We used Giemsa staining with BaOH C-banding  to obserD. australis from the Melbourne Zoo captive population for the assembly. The reference genome sequence was assembled and annotated by Dovetail Genomics; we briefly describe the method here. High-molecular weight DNA was extracted from snap frozen muscle tissue. About 588.2 Gb of PacBio CLR reads were generated with this extract. Contigs were assembled with WTDBG v2.5 (We sampled a single adult male DBG v2.5  with an DBG v2.5  and remoDBG v2.5 . ProximiDBG v2.5 , and HiRDBG v2.5  was usedDBG v2.5  with thede novo and classified using the software package RepeatModeler v2.0.1 (C. hookeri (M. extradentata (T. cristinae (D. australis using both AUGUSTUS software v2.5.5 (Repeat families were identified r v2.0.1 . We furtr v2.0.1  to calcur v2.0.1  with ggpr v2.0.1 . Coding  hookeri , M. extradentata , and T. ristinae  were usee v2.5.5  and SNAPe v2.5.5 . We extre v2.5.5  and intrD. australis into a recently published phasmid phylogeny (de novo assembled transcriptome. We assembled all RNA-Seq reads generated for annotation with Trinity (Ephemera danica, Ladona fulva, Rhodnius prolixus, and Zootermopsis nevadensis (D. australis. We constructed NJ trees of all alignments in R with apetools v5.5 (We placed hylogeny , using a Trinity  and used Trinity  with a 9 Trinity  using thvadensis  and idenvadensis , using tvadensis  in “autools v5.5  and visuols v5.5 . We filtols v5.5 . We usedols v5.5  and Modeols v5.5  to find ols v5.5 .Timema species (e-values.We remapped the long reads used in the original assembly back to the final assembly with minimap v2.21  and esti species  and used species  to identevad104_Supplementary_DataClick here for additional data file.


Unintentional injuries, including traumatic brain injuries (TBI), are the leading cause of pediatric morbidity and mortality in the USA. Helmet usage can reduce TBI incidence and severity; however, the epidemiology of pediatric TBI and helmet use is ever evolving. With lifestyle changes potentially accelerated by the pandemic, we predicted a decrease in helmet utilization with an associated increase in TBI during the pandemic compared to the pre-pandemic period.p = 0.30), as well as the proportion of head injuries from 2018 to 2019 (24.3%) to 2020 (29.3%) (p = 0.07). A significant increase was seen in neurosurgical consultation from 17 (6.5%) in 2018 to 87 (17.7%) in 2020 (p = 0.02). Notably, there was an increase in the percentage of publicly insured patients presenting with injuries from AWHUR during 2020 (p < 0.001); this group also had suboptimal helmet usage.There were 1093 patients that presented with AWHUR injuries from 2018 to 2020 with an annual increase from 263 patients in 2018 up to 492 in 2020. The most frequently implicated mechanisms included bicycles (35.9%), ATVs (20.3%), skateboards (11.6%), scooters (8.3%), and dirt bikes (7.4%). Unhelmeted patients increased from 111 (58.7%) in 2018 to 258 (64.8%) in 2020. There was not a significant difference in the proportion of injuries that were unhelmeted from 38.9% in 2018–2019 to 35.2% in 2020 (This study found an increase in patients presenting with injuries sustained while engaged in AWHUR in relation to the COVID-19 pandemic. Concerningly, there was a trend toward decreased helmet utilization and increased injury severity markers. Further analysis is needed into the communities impacted the most by AWHUR injuries. Unintentional injuries are a leading cause of pediatric morbidity and mortality in the USA CDC . This inParticipation in AWHUR is important for pediatric well-being, but can result in preventable injuries, especially if helmets are not used. Multiple studies have established the efficacy of helmets in reducing the incidence and severity of TBI in these activities  with a gross increase from 104 patients in 2018 up to 252 in 2020. AWHUR injuries grossly increased annually from 263 patients in 2018 to 492 in 2020. Additionally, there were 1.85 injuries per 10,000 children in 2018 up to 2.38 injuries per 10,000 in 2019 and up to 3.43 injuries per 10,000 in 2020, with 1.37  change rate per 10,000 per year. (p < 0.001).A total of 1093 patients presented with injuries from AWHUR over the time frame  (Table p = 0.53). Over the 3-year period, publicly insured patients presented unhelmeted approximately 66% of the time while privately insured children presented unhelmeted 44% of the time (p < 0.001). Over the 3 years, dirt bike injuries presented with a helmeted rate of 79.2%.Helmet use was documented for 833 (76%) of patients; of these, 62.9% of patients were unhelmeted at the time of injury (Table p = 0.50) (Table p < 0.001) (Table p < 0.001). Additionally, 8% of patients who were wearing a helmet required an emergent neurosurgical consultation compared to 24% of patients who presented without a helmet  Table . Of patip = 0.035) (Table p = 0.02).The overall ISS of patients during COVID-19 with a very severe score (> 25) was 3.6% compared to pre-pandemic average of 1.6%. Breaking down the ISS by helmet status over the 3 years, 1.3% of helmeted patients had a score > 25 compared to 3.6% of unhelmeted patients , there was an alarming trend toward an increase in children with a GCS of less than 8 from 2.5% pre-pandemic to 3.3% during the pandemic, although this was not statistically significant. Additionally, finding that approximately 7% of children presenting unhelmeted had a GCS of 12 or less compared to 0.9% of helmeted patients again aligns with our knowledge that helmets can reduce severe brain injury , off-road-vehicles , skiing, snowboarding, ice skating, roller skating, rollerblading, skate boarding, and equestrian sports.Children aged 0 to 18 years old were eligible for the study if they presented to include EDs between March 14 and December 31 of 2018, 2019, and 2020, and they were included in the trauma registry. These time periods were chosen to coincide with the public health state of emergency declared by the state. The registry data are defined by the American College of Surgeons National Trauma Standard Data Set. The main patient qualifications to be included in the registry are: one or more traumatic injuries sustained within the previous 14 days of a hospital encounter; direct hospital admission or transferred from another facility via emergency medical services (EMS) or air ambulance; death in the ED; and presentation as a trauma activation requiring surgical presence per hospital guidelines.Patients were broadly included from the above-mentioned trauma registry based on the following International Classification of Diseases (ICD)—10 codes: S00-S99, T07, T14, and T79.A1-T79.A9 with one of the injuries in the following codes: S00, S10, S20, S30, S40, S50, S60, S70, S80, and S90 , activity type, helmet use, presence of head injury, neurosurgical consultation, and extent of head injury. Head injuries were identified through the Glasgow Coma Scale (GCS) and ICD codes consistent with a head injury. Data were analyzed for each year to compare the same period pre-COVID and to the time frame of the statewide public health state of emergency in 2020. Children who presented with unknown helmet status after review of individual patient charts were included in mechanism analysis but were excluded from the head injury analysis in relation to helmet status.To assess the impact of injuries within the patient community, data was pulled from the 2020 US Census to identify the pediatric population in the 17 counties that comprise the majority (97%) of ED visits at this pediatric healthcare system. This study received approval via the children’s hospital institutional review board (IRB).P < 0.05.For descriptive analyses, patient demographics and clinical characteristics were summarized using means and standard deviations or medians and interquartile ranges for continuous data and counts and percentages for categorical data. Differences in patient characteristics between pre-COVID (2018–2019) and COVID (2020) periods were assessed using chi-square tests (or Fisher’s exact tests). Trends were calculated using linear regression models with logged case counts as the dependent variable over the 3-year time period from 2018 to 2020. The number of injuries due to AWHUR were standardized by dividing our patient catchment population with US Census Data, multiplied by 10,000. The annual rate of change and corresponding to 95% confidence interval (CI) were reverse transformed for interpretation and reported. The different distribution in age, insurance status, injury, and requiring neurosurgical consultation between injuries with helmet use and injuries without helmet use was measured by chi-square tests. All statistical analyses were performed using SAS software  and R (v4.0.2). Statistical significance was set at


Health plans are using 3-tier copayment designs and other methods to control utilization that shifts drug costs to plan members. There is a need to determine the effects of increased member cost sharing on drug utilization and drug costs.To assess the impact of a $10 increase in prescription copayment in a public employer health plan for 2 classes of drugs used for allergic rhinitis.Changes in the number of prescriptions dispensed for 2 therapeutic classes low-sedating antihistamines (LSAs) and nasal steroids (NSs) were examined 1 year prior to and 1 year after copayment increase. Relative price effects were measured as arc price elasticity, the ratio of the percent change in prescription utilization over the percent change in price, an indicator of how responsive patients are to the copayment increase.Of 8,643 continuously enrolled health plan beneficiaries, 2,150 patients (24.8%) received at least 1 NS or LSA during the 2-year period of the study, from January 1, 1998, through December 31, 1999. An average $10 increase in copayment per prescription was associated with no statistically significant change in utilization of combined LSA and NS prescriptions, 2.89 per patient in 1998 and 2.94 in 1999 (P = 0.597). Health plan costs for study drugs, unadjusted for inflation, decreased by 16.3% from $86.86 per patient in 1998 to $72.68 in 1999 (P = 0.004). Health plan costs per patient per month (PPPM) for all drugs for the 2,150 allergic rhinitis patients decreased by 13% from $41.33 PPPM in 1998 to $35.93 in 1999 (Pless than0.001), and health plan drug costs for all 8,643 members decreased by 13% from $14.93 per member per month (PMPM) in 1998 to $12.99 in 1999 (Pless than0.001). The actual average copayment increase was $7.23 (a 41% increase) for LSAs, which was associated with a 14.8% increase in utilization of LSAs and an 11.8% increase in the number of patients using LSAs; the number of LSA prescriptions per patient per year was unchanged at 2.68 in 1999 versus 2.61 in 1998 (P = 0.429). The actual average copayment increase was $10.98 (71%) for NSs, which was associated with an 11.3% decrease in utilization of NSs and a 10.2% decrease in the number of users of nasals steroids in 1999; the number of nasal steroid prescriptions per patient per year was unchanged at 2.05 in 1999 versus 2.07 in 1998 (P = .842). The combined utilization of LSA and NS prescriptions increased by 8.9% following the increase in copayments for these 2 therapeutically interchangeable drugs for allergic rhinitis. LSA prescriptions were less elastic, with an unadjusted arc elasticity of 0.39, while nasal steroid prescriptions were more responsive to the copayment change, with an unadjusted arc elasticity of .0.22.An average $10 increase in patient cost sharing per prescription (46.9% copayment increase) was associated with an increase in combined utilization of 2 drug classes used for allergic rhinitis (LSAs and NSs) but no change in the number of prescriptions per patient. Health plan costs decreased significantly for allergic rhinitis drugs, all drugs used by allergic rhinitis patients, and all drugs used by continuously enrolled health plan members. NSs exhibited a greater arc price elasticity compared with low-sedating oral antihistamines.


Hepatocellular carcinoma (HCC) is a type of primary liver malignancy. Worldwide, HCC is one of the leading causes of cancer-related deaths in adults. Liver cancer is currently the sixth most common cancer worldwide. Many changes in the epidemiology and underlying etiology of HCC have taken place in recent years. In industrialized countries, NAFLD/NASH has been playing an increasingly important role in the development of HCC. Despite major advances in the systemic medical management and availability of immunotherapy, no marked improvement in overall survival time has been found over the past decade.n = 128, n = 148). The median age of diagnosis was 72.5 years (SD 8.6). Patients were on average 2 years younger in the second time period compared to the first (p = 0.042). Alcohol remained the leading underlying etiology of HCC and no statistically significant change was seen over time (p = 0.353). Nevertheless, a clear upward trend in the number of NASH cases was evident over time . Nearly half of the patient population had a normal AFP (<7 µg/L) level at the time of diagnosis . The survival time for HCC patients remained similar between time periods, with a median overall survival time of 20.5 months , despite improvements in management strategies and the availability of new systemic treatments. More advanced-stage HCC cases were documented in the second period . An increased number of HCC patients without liver cirrhosis were identified during the second time period . NASH was the most common underlying etiology in patients without liver cirrhosis (50%) compared to alcohol use in being the primary cause in cirrhotic patients . Conclusion: HCC continues to be an important health concern in our society. The number of HCC patients without liver cirrhosis is steadily increasing, with NAFLD/NASH, due to underlying lifestyle diseases playing an important etiological role. Continued efforts should be made to prevent HCC and to screen at-risk population groups. Preventative strategies and screening techniques should be adjusted in light of the changing epidemiological landscape of HCC, where more focus will have to be placed on detecting HCC in patients without underlying cirrhosis.Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths and remains a major burden on healthcare systems worldwide. The incidence of HCC continues to rise globally, despite preventative efforts being made. Aims: This study aimed to investigate epidemiological changes observed in the etiology and survival outcomes of HCC patients at Klinikum Klagenfurt am Wörthersee between 2012 and 2023. Methods: This was a retrospective, single-center cohort study. Two time-periods (2012–2017 and 2018–2023) were created to enable comparison between the respective intervals. IBM SPSS was used to analyze statistical data. Results: More patients were diagnosed with HCC during the second time period ( Hepatocellular carcinoma (HCC) is a type of primary liver malignancy. The majority (90%) of primary liver cancer cases are attributed to HCC ,2. WorldHCC mainly develops in the setting of underlying liver cirrhosis . HepatitBetween 2010 and 2018, a total of 7146 individuals were diagnosed with HCC in Austria, of which 75% were male and 25% were female . A totalIn Austria, alcohol remains the predominant cause of liver cirrhosis. According to the textbook of alcohol in Austria, the number of hospital admissions for alcohol dependency have however steadily declined over the past decade .The Barcelona Clinic Liver Cancer (BCLC) system is the most frequently used staging tool in clinical practice today. The European Association for the Study of the Liver (EASL) recommends the updated BCLC staging system for the prediction of prognosis and the allocation of appropriate treatment . In the Furthermore, newly established therapeutic options in systemic therapies have been included. Between 2018 and 2019, the IMbrave150 trail compared Sorafenib to the Atezolizumab/Bevacizumab combination and proved a better overall survival rate with the latter treatment strategy .In this study, we aimed to investigate the changes observed in the epidemiology and etiology of HCC at Klinikum Klagenfurt between 2012 and 2023.A rise in NAFLD/NASH leading to HCC has been observed in other developed countries with limited data available on the incidence of NASH within our more rural region . We therWe furthermore aimed to investigate the survival outcome and prognostic parameters in rural HCC patients. We also aimed to investigate whether or not overall survival has improved over time, after immunotherapeutic agents and more TKI therapies became widely available in 2018.Given that the incidence of HCC is rising worldwide, with HCC having a poor prognosis when diagnosed at a late stage, epidemiological data should be used to improve prevention strategies by providing the necessary information in which patient surveillance is lacking.This was a single-center cohort study at Klinikum Klagenfurt am Wörthersee in Austria. Klinikum Klagenfurt acts as a tertiary hospital for the province of Carinthia and serves a large rural drainage area. Data were obtained retrospectively from the hospital’s clinical information software system and the laboratory results were acquired from the hospital’s electronic system. Each patient’s electronic file was reviewed individually in order to obtain the desired datapoints. Data were collected in an Excel database and kept anonymous by allocating numbers to patient names.Several patient characteristics , clinical features , tumor characteristics , laboratory values , and therapeutic treatments for HCC were documented.Two time periods were established to evaluate the changes in epidemiology and survival. The first time period included all patients diagnosed with HCC from January 2012 until the end of 2017 and the second comprised patients diagnosed between the beginning of 2018 until February 2023. In 2018, a marked change in the systemic medical management of HCC started to take place and 2018 was therefore used as the cutoff for comparison. By creating two time periods, we were able to compare changes in etiology and survival outcomes that had occurred over time. We therefore needed to create two time periods in order to comment on whether or not change had occurred. Regarding survival outcome, we first calculated the survival time for the cohort as a whole to provide a general overview; thereafter, we analyzed the changes that occurred over time. Liver cirrhosis was diagnosed by means of radiology , laboratory parameters according to Baveno, Fibroscan, or histologically. First-line treatment was divided into five categories: surgical, which consisted of orthotopic liver transplantation (OLT) and partial hepatectomies; interventional therapy, which included ablation techniques with radiofrequency ablation (RFA) or microwave ablation (MWA); and transarterial chemoembolization (TACE). The fourth category consisted of patients who had received systemic therapy (tyrosine kinase inhibitors or other immunotherapies) as a first-line treatment, and the fifth category included patients who had received best supportive care as first-line treatment. All patients above the age of 18 diagnosed with HCC either histologically by means of a liver biopsy or through multiphase imaging techniques (quadruple-phase CT or contrast-enhanced MRI) were captured in our data collection. Patients with mixed HCC and biliary tract cancers (BTCs) and patients with missing data vital to our study aim were excluded from our study population. n) and percentages (%) and numerical data were presented as the median with the standard deviation range included. Pearson’s chi-square test was applied to analyze categorical data and to determine how likely it was for differences between datasets to occur due to chance. A p-value of <0.050 was seen as statistically significant. Cox regression analysis and the Kaplan–Meier method were used to estimate and analyze survival probability. The IBM SPSS version 25.0  was used to analyze the captured data from the selected patient population. Nominal data were shown as absolute numbers .From January 2012 to February 2023, a total of 285 patients were diagnosed with HCC. A total of 276 patients met the requirements of the inclusion criteria and were considered for further statistical analysis.p = 0.042).The first time period consisted of 128 patients  and the second of 148 patients (53.6%). The incidence rate was 5.9 times higher in males compared to females. In the first time period, the median age of diagnosis was 73 years (SD 8.6) compared to 71 years (SD 8.5) in the second time period (p = 0.005). During the first time period, 22 patients (17.2%) had HCC without underlying liver cirrhosis. This number increased to 47 (31.8%) during the second time period (p = 0.353).Alcoholic liver disease was the most common underlying etiology, with a total of 139 patients (55.6%). Fifty-five patients (22%) had viral hepatitis and forty-three (17.2%) had underlying NASH. A total of 13 patients (5.2%) were classified as “other” . There was no statistically significant change observed in the etiology of HCC between the two time categories (p = 0.199), despite the shift in etiology of the underlying liver disease.Alpha-fetoprotein (AFP) values were documented for 272 patients. A total of 116 patients (42.6%) had unelevated AFP levels (<7 ng/mL) and 156 (57.4%) had a raised AFP (≥7 ng/mL) level at the time of diagnosis. No difference could be observed between the various etiologies. Only 66 patients (24.3%) had an elevated AFP level ≥200 ng/mL, and 56 patients (20.6%) had an AFP level of ≥400 ng/mL. No significant change in AFP levels was observed over time  were classified as Child–Pugh A. n = 112) were classified as BCLC-A at the time of diagnosis. The number of patients diagnosed at the advanced stage BCLC-C increased over time (p = 0.051).With regard to tumor staging, the majority of patients  of HCC patients were staged as ALBI grade 1. Significantly more patients were staged as ALBI grade 1 in the second time period . The median size of the largest space-occupying lesion was 4.5 cm in both time periods. No statistically significant difference in the incidence of portal vein thrombosis, macrovascular invasion, enlarged lymph nodes, or distant metastases was observed over time. There was no noteworthy change in histological grading over time (p = 0.871).Concerning comorbidities, no significant change was seen over time. The mean body mass index (BMI) for all patients was 28 (SD 4.5). The mean BMI of HCC patients at the time of diagnosis did not change over time (p = 0.580). In our patient population, a total of seven orthotopic liver transplantations (OLTs) were performed in the first time period and six in the second (n = 42) of patients underwent surgical treatment (OLT/resection), 16% (n = 45) underwent ablation by means of RFA/MWA, 28% (n = 76) underwent TACE, 27% (n = 75) obtained systemic medical therapy (TKI/IT), and 14% (n = 38) of patients received best supportive care.Regarding first-line therapy for all patients, 15% (p < 0.001). While surgical treatment remained stable over time, there was a significant shift from interventional to systemic treatments between the first and second observation period (p < 0.001), and only 2 (6.3%) patients received systemic drug treatment in the first time period, compared to 16 (55.2%) patients in the second  in the first time period compared to two patients (6.9%) in the second time period). This occurrence cannot be evaluated statistically due to the low number of patients, as surgery is not a standard form of treatment for BCLC-B patients. However, 26 (81.3%) patients received ablative/interventional therapy in the first time period, compared to 11 (37.9%) patients in the second . The median overall survival was 20.5 months, with 21.9 months (95% CI 17–26.8) in the first time period and 20.2 months (95% CI 15.7–24.6) in the second time period (p = 0.902).When patients who had undergone OLTs were removed from the analysis, the median overall survival was 19.4 months (95% CI 15.9–22.9), with no significant change between the two time intervals (p = 0.262). No significant change in survival time for cirrhotic vs. noncirrhotic patients were found between time periods (p = 0.279). The median OS for HCC patients without liver cirrhosis was 22 months (95% CI 8.7–35.7) and those with cirrhosis had an OS of 20.2 months . No difference in OS for alcohol (p = 0.772), viral (p = 0.868), or NASH (p = 0.970) as underlying etiology was found in cirrhotic compared to noncirrhotic patients.Regarding survival and etiology, patients with alcohol as underlying cause had the shortest median OS of 18.1 months (95% CI 14.2–22). Those with viral hepatitis had a median OS of 32.2 months (95% CI 15.9–48.3). Patients with NASH had a median OS of 38.7 months (95% CI 18.4–59) and those with other causes had a median OS of 19.7 months  (see Patients with BCLC-A had a median OS of 34.9 months (95% CI 15.8–53.9), those with BCLC-B had 27 months (95% CI 17.9–36.2), BCLC-C had 10 months (95% CI 7.3–12.8), and BCLC-D a median OS of 1.9 months (95% CI 1.3–2.6) (001) see B.p = 0.805). The OS for BCLC-C in the first time period was 10.4 months (95% CI 5.9–15) and 10 months in the second time period .Looking at patients with early-stage (BCLC-A) HCC and advanced-stage (BCLC-C) separately, OS in the first period for BCLC-A was 33.1 months (95% CI 11.2–55.1) and in the second period 53 months .No significant difference in overall survival for patients with cirrhosis versus non-cirrhosis in BCLC-A and BCLC-C, respectively, was found (p < 0.001) , for Child–Pugh stage B 14.6 months (95% CI 10.1–19), and 2.6 months (95% CI 0.2–5) for Child–Pugh C (001) see A.p ≤ 0.001).Patients classified as Child–Pugh A had a survival time of 28.7 months (95% CI 19.8–37.6) in the first time period compared to 27 months (95% CI 12.8–41.3) in the second time period. Patients with Child–Pugh stage B had a survival time of 15.2 months (95% CI 9.3–21) and 11.8 months (95% CI 0.9–22.7) in the respective time periods and Child–Pugh C a survival time of 1.8 months (95% CI 0–3.6) in the first and 5.6 months (95% CI 0–13.7) in the second time period (p < 0.001) . For patients classified as ALBI grade 2, the median survival time was 19.4 months (95% CI 15.1–23.6) and 6.6 months (95% CI 1–12.2) for ALBI grade 3. There is a significant change over time for survival of the various ALBI grades (001) see C.p < 0.001). Patients with a normal AFP level (<7) had a median survival time of 34.6 months (95% CI 22.8–46.3) and those with a raised AFP above 7 had a 14.6-month median survival time (95% CI 10.3–18.9). Patients with an AFP serum level >7 and ≤200 ng/mL had a median survival time of 20.7 months (95% CI 16.1–25.4). Those with an AFP >200 and ≤400 had a median survival time of 5.2 months (95% CI 3.2–7.2) and those with an AFP ≥ 400 had a 7.3 month (95% CI 4.4–10.3) survival time (p < 0.001). The median survival time for those who fulfilled the up-to-seven criteria (≤7 points) was 33.1 months (95% CI 17.9–48.4), while those with >7 points had a median survival time of 11.8 months  of patients had underlying alcoholic liver disease, 20.1% (n = 41) had viral hepatitis, 9.8% (n = 20) had NASH, and 5.4% (n = 11) of patients were classified as other (p < 0.001).With regards to etiology in noncirrhotic patients, 7 patients (15.2%) were known with alcoholic liver disease, 14 patients (30.4%) had underlying viral hepatitis, 23 (50%) had NASH, and 2 patients (4.3%) were classified as other. In patients with liver cirrhosis, 64.7% , similar to the 40% (n = 83) of cirrhotic patients who were also diagnosed as BCLC-A. 21.7% (n = 15) of noncirrhotic patients were classified as BCLC-B, as was the case with 22% (n = 46) of cirrhotic patients. Of the total, 34.8% (n = 24) was staged as BCLC-C and 1.4% (n = 1) as BCLC-D, which was in clear contrast to cirrhotic patients, where 22% (n = 45) was BCLC stage V and 16% (n = 33) was BCLC-D (p = 0.006).The largest percentage of noncirrhotic patients were staged as BCLC-A (n = 32) of patients without cirrhosis had a raised AFP (≥7) compared to 60.8% (n = 124) of patients with cirrhosis who had a raised AFP level (p = 0.047).Only 47.1%  had mild fibrosis, 24 (34.8%) had moderate fibrosis, and 39 (56.5%) had severe fibrosis. p = 0.149).The median size of the largest space-occupying lesion was 5.3 cm (SD 3.4) in noncirrhotic patients, compared to 4 cm (SD 3.7) in cirrhotic patients (Eleven patients (15.9%) had PVT at the time of diagnosis, nine patients (13%) had distant/extrahepatic metastases, thirteen patients (18.8%) had MVIs, thirteen patients (18.8%) had enlarged lymph nodes (>2 cm), and nine (13%) had SBLs. Patients without liver cirrhosis predominantly (68.1%) had lesions restricted to one lobe of the liver.Apart from arterial hypertension, there was no difference in comorbidities between patient with and without cirrhosis. p > 0.001).Surgical treatments and systemic therapies including immunotherapy were significantly more often performed in noncirrhotic patients, while interventional treatments were more common in cirrhotic patients , NASH was the cause of HCC in half of noncirrhotic patients and a clear upward trend in the number of NASH patients was demonstrated over time . In a laThe nonalcoholic fatty liver disease process starts with a nonalcoholic fatty liver (NAFL), which may worsen and develop into nonalcoholic steatohepatitis (NASH). NASH is characterized by steatosis and accompanied by hepatocyte inflammation, which may include fibrosis . Most noDespite the increase in HCC in noncirrhotic patients, comorbid lifestyle diseases generally known as predisposing factors for the development of NASH did not significantly increase over time. It is, however, important to note that we still had a much higher incidence rate of diabetes in our cohort compared to the general population. Currently, the global incidence of diabetes in adults is around 10.5%, compared to 32% of patients in our cohort . The samWith regard to OS, patients in our cohort survived between 20 to 22 months. We did not observe any further difference in survival between cirrhotic and noncirrhotic patients. The similar OS rate of cirrhotic vs. noncirrhotic patients can be explained by the more advanced BCLC stage of noncirrhotic patients at the time of diagnosis. This finding highlights the lack of surveillance protocols for noncirrhotic patients.AFP remains a common screening parameter used in practice today, but nearly half (43%) of patients did not have a raised AFP (<7 ng/mL) at the time of diagnosis. Furthermore, AFP was only elevated in 47% of the noncirrhotic patients, compared to 60% of cirrhotic patients. Our data are corroborated by a review written by Desai et al., where it was also found that AFP levels were only elevated in 31–67% of noncirrhotic cases, compared to 63–84% in cirrhotic patients . This fiTo improve the early detection of HCC and surveillance of cirrhotic patients, Klinikum Klagenfurt introduced educational programs in the region after becoming an expert primary center for HCC in 2016. Our center therefore played an increasingly important role in facilitating the finding and evaluation of suspicious hepatic lesions and initiating individualized treatment plans. The relatively high rate of Child–Pugh A, BCLC-A, ALBI I, and significantly lower age at the time of diagnosis in the second time period might reflect the successful implementation of improved screening programs. Regarding the treatment of HCC, 2018 marked the year of significant changes in the medical management of HCC after the approval and widespread availability of new immunotherapies within the European Union (EU) . The comThe fact that less patients were staged as BCLC-B and more with BCLC-C in the second time period caused a treatment shift away from interventional therapies and led to an increasing number of patients being treated with systemic therapy. In other words, the concept of treatment-stage migration in BCLC-B caused a higher number of systemic therapies. The retrospective analysis of the treatment modalities in our cohort also revealed a significant difference between noncirrhotic and cirrhotic patients, where systemic treatment was the most common first-line treatment of noncirrhotic patients, compared to interventional treatment in the cirrhotic group. The majority of noncirrhotic patients were staged as BCLC-C, which made them unsuitable candidates for interventional treatment. p < 0.001) [Despite the introduction of the aforementioned novel substances, there was no significant improvement in the median overall survival time between the two time periods. However, OS still improved when compared to data collected in Austria over the past three decades. In a previous study conducted by our working group, regarding the epidemiology of HCC in Austria, patients diagnosed between 1990 and 1999, had a mean survival time of 2.6 months, those diagnosed between 2000 and 2009 had a 5.6-month survival time, and those diagnosed with HCC between 2010 and 2018 had an OS of 9.3 months (The ALBI score represents a biochemical score, including serum bilirubin and albumin levels suggesting overall hepatic function . Our nonUnfortunately, only 13 patients in our cohort received an OLT. This might be due to previous, long waiting lists for liver transplantations, causing patients to progress to more advanced stages of disease while awaiting surgery. Some patients also regrettably continued to consume excessive amounts of alcohol and were therefore excluded from the transplantation list.This study was limited by the small study population and retrospective data collection. Our study was, however, strengthened by the inclusion of objective study parameters, such as laboratory values. Patient data were well documented and easily accessible in the electronic record keeping system. Klinikum Klagenfurt is a tertiary center and the third biggest hospital in Austria. It therefore has a large drainage area and receives referrals from all over the province of Carinthia. The rising incidence of HCC warrants the identification of changes occurring in the epidemiology and underlying etiology of HCC. More patients have been diagnosed without underlying liver cirrhosis in recent years. This group of patients was particularly interesting, as it demonstrated a different etiological pattern compared to HCC patients with underlying liver cirrhosis. NASH secondary to lifestyle diseases played a bigger role in the development of HCC in patients without cirrhosis compared to alcoholic liver disease in cirrhotic patients. Since no improvement in OS was found, one could ask whether there is a need for new screening strategies to prevent a surveillance gap in noncirrhotic patients and improving survival outcomes in the future.


Background Ethiopia is one of the countries in the world with the highest rate of tuberculosis (TB). The aim of this study is to describe the characteristics of the patients with TB admitted to a rural hospital in Ethiopia in terms of both diagnosis and clinical management.MethodsA retrospective descriptive observational study was conducted. Data were collected from patients older than 13 years who were admitted to the Gambo General Hospital for TB between May 2016 and September 2017. The variables studied were age, sex, symptoms, human immunodeficiency virus (HIV) serology, nutritional status, presence of anemia, chest x-ray or other complementary tests, type of diagnosis , or clinical diagnosis), treatment received, outcome, and days of admission.ResultsOne hundred eighty-six patients, aged 13 years and older, were admitted to the TB unit. About 51.6% were female, and the median age was 35 years (interquartile range (IQR) 25-50). Cough was the most frequent symptom on admission (88.7%), and contact with a TB patient was only recognized by 22 patients (11.8%). HIV serology was performed in 148 patients (79.6%); seven were positive (4.7%). About 69.3% met the criteria for malnutrition (body mass index (BMI) <18.5). Most patients, 173 (93%), presented with pulmonary TB and were new cases (94.1%). Patients were diagnosed by clinical parameters in 75% of cases. Smear microscopy was performed in 148 patients, of which 46 (31.1%) were positive, and Xpert MTB-RIF results were only obtained in 16 patients, of which 6 (37.5%) were positive. Chest x-rays were performed in most patients (71%) and were suggestive of TB in 111 (84.1%). The average length of hospital stay was 32 days  13-50.5). Women tend to be younger than men, have more extrapulmonary TB, and were admitted longer. Nineteen patients died during admission (10.2%). Patients who die were more frequently malnourished , tend to be admitted for a shorter time than the survivors and receive more concomitant antibiotic treatment.ConclusionsIn this rural Ethiopian setting, patients admitted to the hospital for TB are often malnourished (67.1%), the main presentation is pulmonary, mortality is one in 10 admissions and very often receive antibiotics in association with TB treatment (40%). Ethiopia is a country with a significant burden of poverty, and its economy is still based primarily on agriculture and livestock, with more than 70% of the population living in rural locations . It is oGambo General Hospital (GGH) is a rural (tertiary) facility located about 250 km south of the capital that has a unit dedicated to in-patients with TB. It also has a laboratory for the microbiological diagnosis of TB by fluorescence microscopy as well as a basic radiology unit. Since 2015, it is possible to send samples to the reference laboratory for the Xpert MTB/RIF  assay following national protocols. The aim of this study was to describe the characteristics of the patients with TB admitted to this hospital in terms of both diagnosis and clinical management.A retrospective descriptive observational study was conducted. Data were collected from patients older than 13 years who were admitted to the GGH for TB between May 2016 and September 2017. The inclusion criteria were patients older than 13 years with a diagnosis of TB who were admitted to the GGH during the study period. The rest of the admitted patients were excluded.The variables studied were age, sex, symptoms, HIV serology, nutritional status, presence of anemia, chest x-ray or other complementary tests, type of diagnosis , treatment received, outcome, and days of admission.All data collected were double-checked for completeness. They were entered into a database  and exported to the statistical program SPSS  for analysis. Continuous variables were expressed as the median and interquartile range (IQR), and qualitative variables as value and percentage. The Chi-square test and Mann-Whitney U test were used to compare data where appropriate, interpreting results using odds ratios (OR) and 95% confidence intervals (95% CI). A bivariate analysis was performed to identify variables associated with the outcome of death and with TB by gender, according to the chronological and anatomical classification of TB patients.The Research Committee and Ethics Review Board of the Gambo General Rural Hospital, Ethiopia (Code GGRH-241-18) and the Research Ethics Committee of the Fundación Hospital Universitario Jiménez Díaz, Spain (Code EO187-18_FJD) approved the study. Due to its retrospective nature, the need for informed consent from patients, parents, or legal guardians was waived.Clinical characteristicsBetween May 2016 and September 2017, 186 patients aged 13 years and older were admitted to the TB unit of GGH. Of these, 90 were male (48.4%) and 96 were female (51.6%), with a median age of 35 years (IQR 25-50). Cough was the most frequent symptom on admission (88.7%), and contact with a TB patient was only recognized by 22 patients (11.8%). Human immunodeficiency virus (HIV) serology was performed in 148 patients (79.6%); seven were positive (4.7%)  in 166 patients, of whom 115 (69.3%) met the criteria for malnutrition (BMI<18.5). Likewise, mid-upper arm circumference (MUAC) was also used in 120 patients, where 100 (83.3%) met malnutrition criteria (<22 cm for women and <23 cm for men).Regarding the location of TB and following the 2012 and 2017 national protocols, most patients, 173 (93%), presented with pulmonary tuberculosis (PTB) (any patient with pulmonary infiltrates). Most of the patients were new cases, 175 (94.1%), compared to only 11 patients (5.9%) classified as pre-treated .Diagnostic processPatients were diagnosed by clinical parameters  in 75% 139/186) of cases and 25% (47/186) by smear or Xpert. Smear microscopy was performed in 148 patients, of which 46 (31.1%) were positive, and Xpert MTB-RIF results were only obtained in 16 patients, of which 6 (37.5%) were positive (Table 39/186 ofChest x-rays were performed in most patients (71%), and they were suggestive of TB in 111 (84.1%), with patterns such as uni- or bilateral infiltrates, miliary pattern, and cavities. In 22 (16.6%) patients, no compatible patterns were found .OutcomesThe average length of hospital stay was 32 days  13-50.5). One hundred and fifty patients (80.6%) were discharged for further follow-up at their health posts; 19 patients died during admission (10.2%); 10 cases (5.4%) were transferred to another hospital for several reasons; and seven patients (3.8%) requested voluntary discharge or disappeared from the hospital without prior notice  as admission coincides with the intensive treatment phase; there were 11 patients who received two drugs because they were in the second phase of treatment (5.9%), and only one did not receive any drugs at all. Other concomitantly administered drugs were antibiotics in 81 patients (43.5%), the most frequent being ceftriaxone in 72 patients (38.7%), vitamin complexes in 43 patients (23.1%), corticosteroids in seven patients (3.8%), non-steroidal anti-inflammatory drugs (NSAIDs) in 44 patients (23.7%), diuretics in 18 patients (9.7%), and transfusions in three patients (1.6%).Analysis by gender and outcomeWomen have a greater tendency to present extrapulmonary TB (EPTB)  and especially lymphadenopathies . They tend to be younger than men . A significant difference was also observed in the duration of admission, with women staying longer  . They also tend to be admitted for a shorter time than the survivors  and receive more concomitant antibiotic treatment .According to the temporal classification of patients Table , pre-treThere are few published data on TB patients admitted to hospitals in Ethiopia. Most of the publications are on patients co-infected with HIV or with multidrug-resistant TB (MDR-TB). There is also scientific literature on patients in directly observed treatment (DOT) programs in Ethiopia ,5. Our pOur HIV-positive rate is 4.7%, which is lower than in other series in Oromia (where about 30% of TB patients are HIV positive) and in other areas of Ethiopia, with rates around 19-26%. In addition, in our study, the HIV test was not performed in 20.4% of the patients, while in other studies it was not performed in only 4% -10. PerhRegarding TB location, most of the patients in our study were classified as PTB 93%), compared to 7% of EPTB patients. These rates are much higher than those published in other series, both in general hospitals, referral hospitals, and in patients in the DOTs program 14-57%) ,9,11,12.3%, compa-57% ,9,1The percentage of pre-treated patients in our series is low compared to other studies in referral hospitals (5.9% vs. 12.1%) , but simWe obtained a mortality rate of 10.2%, but in different studies , it variWe have found a high rate of malnutrition (61% of patients). Poor nutritional status is known to be closely related to TB. Some Ethiopian series describe rates of around 28-40% ,20, whilAlso relevant is the number of antibiotics used concomitantly. About 43% of patients received antibiotics along with anti-tuberculosis treatment, and two different antibiotics were used in 10% of them. It is difficult to compare these data with other series, as there are no similar data published. The WHO has published guidelines for the appropriate use of antibiotics, as it has been established that 30% of the antibiotics administered could be used inappropriately . In EthiOur study has limitations related to the difficulty of obtaining data from non-digitized medical records in low-income countries. However, this study is unique because it describes the population admitted to a TB unit in a rural hospital and its clinical characteristics, as well as the diagnostic process, treatment provided, and outcome. Knowing these peculiarities can help to implement specific programs for these hospitalized patients, reinforcing nutritional treatment and adjusting and improving the use of antibiotics.In this rural Ethiopian setting, patients admitted to the hospital for TB are often malnourished (67.1%), the main presentation is pulmonary, mortality is one in 10 admissions, and they very often receive antibiotics in association with TB treatment (40%). Knowing these peculiarities can help to implement specific programs for these hospitalized patients, reinforcing nutritional treatment and adjusting and improving the use of antibiotics (antibiotic stewardship).


Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins, viz. Cry1A.105 and Cry2Ab2, is a biotechnological option being considered for the management of the major corn pest in Indonesia, the Asian corn borer  (Lepidoptera: Crambidae)). As a part of a proactive resistance-management program for MON 89034 corn in Indonesia, we assessed the baseline susceptibility of field-collected populations of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins. Dose–response bioassays using the diet-dipping method indicated that the lethal concentration (LC50) values of Cry1A.105 and Cry2Ab2 in 24 different field populations of O. furnacalis ranged from 0.006 to 0.401 µg/mL and from 0.044 to 4.490 µg/mL, respectively, while the LC95 values ranged from 0.069 to 15.233 µg/mL for Cry1A.105 and from 3.320 to 277.584 µg/mL for Cry2Ab2. The relative resistance ratios comparing the most tolerant field populations and an unselected laboratory population were 6.0 for Cry1A.105 and 2.0 for Cry2Ab2 based on their LC50 values. Some field populations were more susceptible to both proteins than the unselected laboratory population. The LC99 and its 95% fiducial limits across the field populations were calculated and proposed as candidate diagnostic concentrations. These data provide a basis for resistance monitoring in Bt Corn and further support building resistance-management strategies in Indonesia.Genetically modified MON 89034 corn ( Ostrinia furnacalis (Guenée), is one of the major pests of corn (Zea mays Linnaeus) in Southeast Asia, including the Indonesian archipelago, Vietnam, the Philippines, and China [O. furnacalis infestation in each growing season in tropical areas with 24–30 total development days for each generation. Damage from O. furnacalis occurs not only in the stem of corn plants but also in the whorl, tassel, and ears [O. furnacalis egg masses was as high as nine egg masses per plant in fields in Sleman, Yogyakarta. A recent study (2018–2019) in Lampung and Central Java showed that O. furnacalis infestations were present in as many as 95% of corn plants with an average of four holes per stalk and gallery lengths of 4–6 cm [O. furnacalis larva boring per plant during V10, R1, or R2 resulted in grain yield losses of 4.94%, 4.56%, or 3.76%, respectively [The Asian corn borer, nd China . Areekulnd China  and Camand China  reportedand ears . In vegeand ears  reportedf 4–6 cm . This lestudy 201–2019 in O. furnacalis in Indonesia and other countries in Southeast Asia [O. furnacalis [Bacillus thuringiensis (Bt) insecticidal proteins have been commercialized and are widely grown in corn-growing countries, such as the United States, Brazil, Argentina, Canada, South Africa, the Philippines, and Vietnam for the successful management of corn stalk borers, corn ear feeders, and fall armyworm (Spodoptera frugiperda (J.E. Smith)) [Bt corn technologies are currently in the process of registration in Indonesia [Bt proteins, Cry1A.105 and Cry2Ab2, that are highly effective against key lepidopteran corn pests [O. furnacalis and other corn lepidopteran pests [S. frugiperda in the Philippines [The economic damage on corn indicates the need for effective and ecologically sound management practices for ast Asia ,11, wherast Asia ,12,13. Crnacalis . Genetic Smith)) ,15,16. Sndonesia , includirn pests ,19,20. Man pests ,22. In Slippines .Bt crops [Bt proteins with an independent mode of action significantly reduces the risk of resistance development in populations of the target pests [Bt proteins of different mechanisms of action targeting O. furnacalis, its inherent resistance risk is likely lower than that for single-gene Bt plants [O. furnacalis populations to Cry1A.105 and Cry2Ab2 proteins after the introduction of MON 89034 in Indonesia. Establishing the baseline susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 is important for resistance-monitoring programs in Indonesia. The assessment of the baseline susceptibility would not only allow the assessment of the natural variation among field populations but can also be used to document shifts in susceptibility likely resulting from selection for resistance [O. furnacalis populations. The goals of this study were to establish the baseline susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins in Indonesia and to estimate Cry1A.105 and Cry2Ab2 concentrations to be used as candidate diagnostic concentrations to monitor the development of resistance in O. furnacalis in Indonesia.The development of resistance in populations of the target pests poses the risk to the sustainability of Bt crops ,25,26. Iet pests ,28. Becat plants . Neverthsistance ,31. We hO. furnacalis collected from six provinces in 2013–2015 showed variation in susceptibility to Cry1A.105  of the 24 field populations relative to the laboratory population were 0.1–6.0 based on the LC50 values  did not overlap [Populations of ry1A.105 . The LC50 values . Some fi overlap ,33.O. furnacalis collected from six provinces in 2013–2015 also showed variation in susceptibility to Cry2Ab2, as evidenced by the 0.1–2.0 RR values based on the LC50  did not overlap [Populations of the LC50 . The LC5 overlap .O. furnacalis LC99 value for Cry1A.105 was significantly higher than its LC95 value as indicated by non-overlapping lower and upper limits of the 95% FL  as the candidate diagnostic concentrations: 13.240 (6.716–33.831) µg/mL for Cry1A.105 and 127.320 (46.616–676.792) µg/mL for Cry2Ab2. These concentrations need to be tested with several field populations for further validation, followed by the selection of one diagnostic concentration for each protein to be used in monitoring programs.The e 95% FL . HoweverO. furnacalis to Cry1A.105 and Cry2Ab2 before the introduction of MON 89034 corn and to provide benchmark data for future comparison to detect susceptibility shifts. In this study, the baseline susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 was established based on the populations collected from major corn-producing provinces in Indonesia.There is a need to develop a robust insect resistance-management (IRM) strategy for MON 89034 in Indonesia to prolong its durability in the field and, thus, delay the development of practical resistance once the event is approved for cultivation ,34,35. BBt proteins before the onset of resistance have been reported among geographically distinct populations in many different species of insects attacking corn [O. furnacalis, several studies have been conducted using five proteins, i.e., Cry1Ab, Cry1Ac, Cry1F, Cry1A.105, and Cry2Ab2. Field-collected populations of O. furnacalis in Vietnam differed in susceptibility to Cry1Ab by up to three-fold, which was reflective of natural variability among the 11 populations used in the study [O. furnacalis in China [50 and LC95, respectively, were demonstrated. Furthermore, Alcantara et al. [Differences in susceptibility to ing corn ,36,37,38he study . In Chinhe study , and in he study . Contrasin China , 80-folda et al.  reportedO. furnacalis field populations to Cry1A.105 and Cry2Ab2 than were reported in the Philippines. However, direct comparison between these two studies (diet dipping vs. diet overlay) was not possible since different bioassay procedures were employed. Differences in the susceptibility of O. furnacalis to these two proteins may represent natural variation among populations because transgenic corn has not yet been planted commercially in Indonesia, and commercial Bt formulations for controlling O. furnacalis are not commonly utilized by Indonesian farmers [O. furnacalis to Cry1Ab [Helicoverpa armigera to Cry1Ac (67-fold) in India [We reported higher levels of variation in the susceptibility of  farmers . A high o Cry1Ab  and in Hin India . In addiin India ,44. TherO. furnacalis in Indonesia , two on Sumatra Island (North Sumatra and Lampung), and the province of Gorontalo on Sulawesi  layered with wetted filter paper to maintain the freshness of the eggs. Newly hatched larvae and bigger larvae were transferred individually into clear plastic containers with a screw cap  containing an artificial diet (ca. 5 g) [Ostrinia furnacalis populations collected from different locations were reared in separate cups, trays, and cages to avoid contamination between populations. Additional details are provided below in “Insect-Rearing Procedure”.ca. 5 g)  to minimO. furnacalis populations were reared using a similar artificial diet and standardized laboratory procedures [The field-collected and laboratory ocedures . Larvae 2 generation O. furnacalis neonates and a few using the F1 or F3 generation. The F3 generation was used when neonates of F2 was not sufficient to do the whole bioassays. The larvae of F2 and F3 were used if the F1 neonates in uniform age were inadequate for the conduction of the bioassay. Bioassays were carried out by following the diet-dipping procedure described by Trisyono et al. [O. furnacalis larvae were transferred individually into two plastic cups containing the treated or control diet (5 larvae per cup). Each treatment was replicated 3–5 times. In the Cry1A.105 bioassays, 10 concentrations from 0.002 to 48 µg/mL Cry1A.105 (three-fold dilutions) were tested to determine LC50 and LC95 of 24 field-collected O. furnacalis populations and the laboratory population. For Cry2Ab2, 10 concentrations ranging from 0.0007 to 15.5 µg/mL (three-fold dilutions) and the control were used for all bioassays to determine LC50 and LC95 against the same F1–F3 of field-collected and laboratory populations. Based on the results from the preliminary bioassays, the concentrations used for Cry1A.105 and Cry2Ab2 were different due to varying response of O. furnacalis to each protein. The selected concentrations based on the preliminary test were expected to result in larval mortality ranging from 2% to 98%. Prior to the actual assays, preliminary tests of both proteins were carried out using 10 newly hatched larvae per concentration in three replicates. Larvae were exposed to the treated or control diet continuously, and observed mortality was recorded 7 days after they were placed in the plastic cups containing the artificial diet.The proteins (Cry1A.105 and Cry2Ab2) were provided by Bayer CropScience, St. Louis, MO, USA. Bioassays were carried out using mostly Fo et al.  by dippi50 and LC95 values and their 95% fiducial limits (FLs) for each protein and population. The relative resistance ratios (RR) were calculated by dividing the values of LC50 of field populations by that of the laboratory population [95 and LC99 and their lower and upper limits for the Cry1A.105 and Cry2Ab2 proteins were determined from pooled baseline concentration–mortality data (24 field-collected populations) using the program PoloJr, and the estimated LC99 and its lower and upper limits of 95% were proposed as candidate diagnostic concentrations for each protein, as they killed 99% of the susceptible population [Probit analysis  was carrpulation . The LC9pulation ,53.


The relative ellipsoid zone reflectivity (rEZR) has been proposed as an innovative biomarker for photoreceptor integrity. This study evaluates the rEZR in macular telangiectasia type 2 (MacTel) eyes of different disease stages.The mean rEZR  was analyzed for an entire spectral domain optical coherence tomography volume scan  and for each subfield of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid (topographic) in patients with MacTel and controls. MacTel disease severity was classified according to Gass and Blodi.P < 0.001), the inferior inner , the nasal inner , and in the superior inner subfield of stage 4/5 .Linear mixed-model analysis of 145 eyes of 74 patients and 50 eyes of 25 controls revealed globally lower, yet not statistically significant, rEZR values in MacTel eyes. Topographically, most pronounced decreases were found in stages 3 and 4/5 for the temporal inner (coefficient estimates [CEs] = −25.4  and −34.1  AU, both: The rEZR showed association with disease severity and the predilection area of MacTel. Given the current understanding of the pathophysiological concept of MacTel, these findings underscore the value of the rEZR as a potential novel biomarker for outer retinal integrity. Longitudinal studies are demanded to better characterize its value as a biomarker for early photoreceptor alterations and disease progression in MacTel. In addition, functional complexes between Müller cells and retinal photoreceptors become apparent in SD-OCT imaging as the first hyper-reflective band and is named the external limiting membrane (ELM). The ratio of the EZ to the ELM reflectivity, termed the relative EZ reflectivity (rEZR), has recently been proposed as an auspicious biomarker for outer retinal integrity and photoreceptor health.The purpose of this study is to assess the rEZR within the pathophysiological concept of MacTel with regard to disease staging and its spatial predilection of structural alterations in the retina as well as to evaluate the rEZR in MacTel eyes without manifest EZ loss.www.mactelresearch.org), a multinational natural history study on MacTel. Inclusion criteria were a confirmed MacTel diagnosis and retinal imaging of sufficient quality. Exclusion criteria included the presence of confounding ocular conditions, such as AMD and diabetic retinopathy, as well as optic media opacities. Controls were identified after a comprehensive ophthalmic examination. If both eyes of one participant fulfilled the inclusion criteria, both eyes were included. This study was approved by the local ethics committee at the University Hospital Bonn  and adhered to the tenets of the Declaration of Helsinki and written informed consent was obtained prior to data acquisition.For this cross-sectional case-control prospective study, patients with MacTel and controls were selected from a single center cohort at the Department of Ophthalmology, University of Bonn, Germany. Patients were included between October 2016 and November 2022 in the context of the Natural History and Observational Registry study , a slit-lamp and dilated fundus examination, and retinal imaging. Following pupil dilatation with tropicamide 0.5% and phenylephrine 2.5% eye drops, retinal imaging was performed including 55 degrees color fundus photography , combined confocal scanning laser ophthalmoscopy  and SD-OCT . The 30 degrees × 25 degrees grid was used to allow for rEZR assessment within and beyond the MacTel area.MacTel disease stages were classified according to Gass and Blodi by CFP assessment.https://github.com/bisselma/relEZIquantification).,,The rEZR, defined as the EZ/ELM reflectivity ratio, was determined, as previously described, in reflectivity profiles in the raw SD-OCT image files using a semi-automated approach using MatLab . Segmentation coordinates, exported as XML files, were superimposed to the OCT raw images  and used for straightening of each OCT B-scan along with the coordinates of the Bruch's membrane enabling accurate rEZR determination even in eyes with pronounced posterior pole curvature. Within every single OCT B-scan, the rEZR data was obtained at adjoining regions of interests (ROIs) in corresponding reflectivity profiles , see To assess the rEZR both globally (i.e. within the entire SD-OCT volume scan) and topographically (i.e. focusing the “MacTel area”), the Early Treatment Diabetic Retinopathy Study (ETDRS) grid was used to determine the mean rEZR within each of the nine ETDRS subfields.P values <0.005 were considered significant (due to adjustments for multiple testing by Bonferroni-correction for 10 subfields). In order to better understand the cohort's data structure, a refined analysis of the study cohort was further performed and aimed in evaluation of the inter-eye correlation of cases and controls with bilateral inclusion as well as of the heterogeneity of the data set. The inter-eye correlation was assessed in determining Spearman's correlation coefficient r, which was calculated for the mean rEZR of the total SD-OCT volume raster scan of each participant with both eyes included. Further, heterogeneity of the data was evaluating by comparing the variance as well as the range of the rEZR values between patients with MacTel and controls.Patient characteristics are presented as absolute and relative frequencies for categorical variables and as means with standard deviations (SDs) for continuous variables. To investigate the primary research question of differences  between rEZR values in MacTel and control eyes, linear mixed-effects models were used. These accounted for possible correlations between measurements taken from the same patient and/or eye . Differences in the mean rEZR (arbitrary unit [AU]) between MacTel and control eyes were assessed (i) for the total SD-OCT volume raster scan  and (ii) for the individual subfields (topographic assessment) of the ETDRS grid. Additionally, in eyes with MacTel, we considered retinal disease stages according to Gass and Blodi to investigate the association between MacTel disease severity and rEZR. Linear mixed-effect models were adjusted for age and eccentricity, entering the model as a spline (B-spline of degree 2). The P values <0.05 were considered significant. The analyses were performed with the software R, version 4.1.2, using the package lme4.For a refined characterization of the rEZR as a potential biomarker, we conducted an exploratory subgroup analysis of MacTel eyes which did not show manifest EZ loss. Here, A total of 195 eyes of 99 study participants  were included. Out of those, 145 eyes of 74 patients  were diagnosed with MacTel, whereas 50 eyes of 25 participants  were healthy.MacTel staging resulted in 9 eyes (6.2%) of 9 patients with MacTel being classified as stage 1, there were 49 eyes (33.8%) of 33 patients as stage 2, there were 59 eyes (40.7%) of 43 patients as stage 3, there were 25 eyes (17.2%) of 20 patients as stage 4, and 3 eyes (2.1%) of 3 patients as stage 5, respectively. A high level of inter-reader agreement was reached, as readers 1 and 2 agreed in 140 of 145 eyes of 96.5% and a third reader was only required to arbitrate in the remaining 5 cases (3.5%). Out of the 145 MacTel eyes, manifest EZ loss was detectable in 102 eyes (70.3%) of 63 patients. In detail, 4 of the 9 stage 1 eyes demonstrated manifest EZ loss , 29 of 49 stage 2 eyes , 41 of 59 stage 3 eyes , and 25 of 25 stage 4 eyes , respectively. The 3 eyes classified as stage 5 also showed manifest EZ loss.r = 0.164 for controls and r = 0.134 for patients with MacTel indicating a minor inter-eye correlation. The variance of rEZR values for MacTel eyes was more than five times higher than that of controls (24.15 vs. 135.61 AU). Consistent with these findings, the range of rEZR values in MacTel eyes was wider (on average 9.14 AU) than in controls (on average 8.40 AU). Accordingly, MacTel eyes tend to have a higher degree of heterogeneity and more extreme values than controls. Bilateral data were available from every control patient and from 71 out of 74 patients with MacTel.Descriptive analysis revealed a median (first quartile and third quartile) rEZR per SD-OCT volume scan of 36.9 (11.6 and 79.2) AU in MacTel eyes and of 56.9 (17.7 and 111.1) AU in control eyes. Regarding the refined analysis of the cohort's data structure, Spearman's correlation coefficient was assessed for the mean rEZR per volume raster scan in all 25 included controls and in 71 out of 75 patients with MacTel. It was revealed to be n = 58, 36 patients), 32.1 (7.6 and 70.7) AU in stage 3 eyes  and 28.5 (8.4 and 60.1) AU in MacTel stages 4/5 eyes . Given the inhomogeneous sample size across MacTel disease stages, results of MacTel diseases stage 1 and stage 2 will be reported in the following as one group (MacTel stages 1/2) as well as of stage 4 and stage 5 (MacTel stages 4/5). Across MacTel disease stages, the median rEZR per SD-OCT volume scan was revealed to be 39.5 (15.4 and 82.9) AU in MacTel stages 1/2 eyes (P = 0.141). Considering MacTel disease staging, this rEZR decrease was more pronounced, as MacTel severity increased . Linear-mixed models, accounting for patients’ age and the topographic dependence of the 36,626 ± 9109 (mean ± SD) rEZR data points within the SD-OCT raster scan, were applied for refined analysis of the rEZR across different MacTel disease stages. Compared to controls, global assessment revealed a decreased rEZR of all MacTel eyes , inferior inner (P < 0.001), and nasal inner (P = 0.004) ETDRS subfield with CEs of −23.2 (−35.6 and −10.8) AU, −25.2 (−39.4 and −11.1) AU and −19.7 (−33.1 and −6.3) AU, respectively. Further topographic results are given in The rEZR decrease of MacTel eyes showed a topographic dependency being more pronounced in the direct parafovea compared to the foveal or more eccentric retinal region. Biggest rEZR differences between controls and MacTel eyes across all disease stages were found in the temporal inner (P = 0.035) in MacTel stages 1/2 eyes, of −25.4 (−38.2 and −12.6) AU (P < 0.001) in stage 3 and of −34.1 (−48.7 and −19.6) AU (P < 0.001) in stages 4/5, respectively. In the inferior inner subfield, the rEZR was decreased with a CE of −13.2 (−28.3 and 1.9) AU (P = 0.087) in stages 1/2 MacTel eyes, of −29.9 (−44.6 and −15.1) AU (P < 0.001) in stage 3 eyes and of −35.3 (−52.1 and −18.5) AU (P < 0.001) in stages 4/5 eyes compared to healthy eyes. Analyses of the nasal inner and the superior inner subfield also showed decreased rEZR values with a CE of −10.0 (−24.5 and 4.5) AU (P = 0.177) and −6.1 (−21.5 and 9.3) AU (P = 0.436) in stages 1/2 eyes, of −21.5 (−35.2 and −7.4) AU (P = 0.003) and −17.2 (−32.3 and −2.1) AU (P = 0.025) in stage 3 eyes as well as of −31.6 (−47.6 and −15.6) AU (P < 0.001) and −25.0 (−42.0 and −7.9) AU (P = 0.004) in MacTel eyes classified as stages 4/5. Detailed results of the impact of MacTel disease staging and retinal topography on the rEZR are presented in The associations of MacTel disease staging and the retinal region with the rEZR remained apparent when considering both simultaneously. The mean rEZR of each ETDRS subfield was shown to be more decreased with advancing disease stages and this decrease, again, was more pronounced in the pericentral ETDRS subfields. Compared to healthy eyes, the rEZR in the temporal inner subfield was decreased with a CE of −14.1 (−27.2 and −1.0) AU  showed no signs of manifest EZ loss. Five eyes  were previously classified as MacTel stage 1, 20 eyes  as stage 2, and 18 eyes  as stage 3. Descriptive analysis showed a median rEZR of 29.5 (9.0 and 65.7) AU in MacTel eyes without EZ loss.P = 0.412) in the global SD-OCT assessment. Topographic analysis showed most pronounced rEZR decreases in the temporal inner  and in the inferior inner  ETDRS subfield. Linear-mixed models revealed that all included MacTel eyes without manifest EZ loss exhibited a decreased rEZR with a CE of −5.2 (−17.6 and −7.2) AU (P = 0.021) and −28.4 (−48.7 and −8.1) AU (P = 0.006), respectively.With regard to disease staging of the included MacTel eyes without manifest EZ loss, topographic analysis revealed the rEZR decrease to be more pronounced with advancing disease staging in all of the four inner ETDRS subfields. Herein, assessed stage 3 MacTel eyes without manifest EZ loss, exhibited a statistically significant rEZR decrease in the temporal inner and the inferior inner ETDRS subfield indicating CEs of −21.2 (−39.3 and −3.2) AU  of different disease stages, please see This is the first study evaluating the rEZR, a potential novel biomarker for outer retinal integrity, in patients with MacTel, demonstrating lower rEZR values in MacTel eyes compared to controls. This difference was revealed to underlie a spatial dependence with pronounced differences in the predilection area of MacTel which, moreover, were also apparent in eyes without manifest EZ loss. Additionally, the rEZR was shown to be associated with disease severity indicated by lower rEZR values in advanced MacTel stages.,Given current assumptions of photoreceptors’ mitochondria and their junctional complexes with Müller cells being the source of the EZ and ELM reflectivity signal in SD-OCT imaging, respectively, their assessment has become a well-established indicator for photoreceptors’ integrity and outer retina degeneration in MacTel.P < 0.001). In the global analysis, however, the rEZR decrease in MacTel eyes was revealed to be both less pronounced (CE of −7.7 [−17.8 and 2.5] AU) and not statistically significant (P = 0.141). These findings are in line with previous studies highlighting the topographical predilection of retinal alterations in MacTel being mostly located temporal and inferior to the fovea, in the so-called “MacTel area.”,–This study demonstrates in the topographical assessment that across all included MacTel eyes the rEZR decrease was most and significantly pronounced in the temporal inner and inferior inner ETDRS subfield with a CE of −23.2 (−35.7 and −10.8) AU and of −25.2 (−39.4 and −11.1) AU  warranting therefore high quality and comparability. Additionally, it needs to be mentioned that the rEZR was determined as the ratio of the EZ to ELM reflectivity (see Methods section for further details). As explained, the ELM is assumed to represent junctional complexes between Müller cells and photoreceptors, which can both alter during the disease process of MacTel.,–Several limitations need to be considered in the interpretation of this study. Although this is the first study evaluating the rEZR in MacTel, other concurrent SD-OCT based features of MacTel have not been considered here. Second, this study aimed in only assessing structural image data of patients with MacTel and does therefore not include any analysis of visual function and its correlation with the rEZR. Further, because analyses were performed on a cross-sectional image data set, further longitudinal studies are needed to evaluate the rEZR's prognostic value for disease progression. However, a strength of this study is the prospectively acquired image data set in context of an international multicenter natural history study (In conclusion, this is the first study assessing the rEZR in MacTel revealing a decrease of the rEZR being associated with disease staging and which was spatially more pronounced in the parafoveal predilection area of MacTel. These findings are in line with the current understanding of the MacTel pathophysiology supporting the rEZR to be a potential innovative SD-OCT biomarker for outer retinal integrity and MacTel severity. This study warrants further analyses, including a refined structure-function-correlation and evaluation of the rEZR's prognostic value for disease progression in MacTel.