Borrelia genus. Here, we report on the widespread infection by a non-described Borrelia species in passerine-associated ticks in tropical rainforests of French Guiana, South America. This novel Borrelia species is common in two tick species, Amblyomma longirostre and A. geayi, which feed on a broad variety of neotropical mammal and bird species, including migratory species moving to North America. The novel Borrelia species is divergent from the LD and RF species, and is more closely related to the reptile- and echidna-associated Borrelia group that was recently described. Genome sequencing showed that this novel Borrelia sp. has a relatively small genome consisting of a 0.9-Mb-large chromosome and an additional 0.3 Mb dispersed on plasmids. It harbors an RF-like genomic organization but with a unique mixture of LD- and RF-specific genes, including genes used by RF Borrelia for the multiphasic antigen-switching system and a number of immune-reactive protein genes used for the diagnosis of LD. Overall, our data indicate that this novel Borrelia is an intermediate taxon between the LD and RF species that may impact a large host spectrum, including American mammals. The designation “Candidatus Borrelia mahuryensis” is proposed for this species.Lyme disease (LD) and relapsing fevers (RF) are vector-borne diseases caused by bacteria of the  Borrelia (Spirochaetes) are the causative agents of major vector-borne diseases including Lyme disease (LD), the most important tick-borne disease in the northern hemisphere, and relapsing fevers (RF), which are transmitted by ticks and lice worldwide2. While RF epidemics occurred repeatedly in past centuries2, LD is an expanding infectious disease with more than 300,000 new human cases each year in the United States4. Most LD and RF Borrelia species are maintained in enzootic cycles and their presence in humans and domestic animals is incidental to their usual wildlife reservoir hosts5.Bacteria of the genus Borrelia species form two separate sister groups7, and are sometimes referred to as two sister genera by various authors9. LD and RF species were usually thought to be the only two groups within Borrelia. However, recent surveys have uncovered new Borrelia species and strains that do not fall into the LD or RF groups21. They form a third group, sometimes referred to as “reptile group” (REP), encompassing only two designated species, B. turcica and “Candidatus Borrelia tachyglossi” (B. tachyglossi hereafter), and a few strains not taxonomically described21. The only two genomes sequenced to date confirmed that these Borrelia are substantially different from the LD and RF Borrelia groups19. Members of the third Borrelia group have been detected worldwide, but not in Western Europe21. They have been detected in hard ticks , which are associated mostly with reptiles, but also with echidna21. Recently, new strains of unknown Borrelia species, closely related to the third Borrelia group, were identified in ticks feeding on ground-dwelling birds (for immature stages) and mammals (for adult stages) in Brazil16, Argentina17, and the United States18. However, currently, we know little about these new American Borrelia strains: Only a small DNA fragment of the flagellin flaB gene has been used as an exclusive marker for their identification18. Unfortunately, this short gene fragment is inadequate for inferring their phylogenetic proximity with known Borrelia species since the inner tree topology remains poorly resolved at many nodes because of insufficient sequence polymorphism18.The LD and RF Borrelia group, in passerine-associated ticks from tropical rainforests of French Guiana. This remote territory is a vast equatorial land located on the north-east coast of South America, mostly covered by dense rainforests and with a low density of human population. This novel Borrelia species is common in two tick species feeding on a variety of mammal and bird species. We further cultivated this bacterium and sequenced its complete genome, which showed that this novel Borrelia is intermediate between LD and RF Borrelia, sharing common features with both of them.In this study, we describe a novel species, belonging to the third n = 274), with only few nymphs (n = 16), and no adults , the wedge-billed woodcreeper, Glyphorynchus spirurus , and the crimson-hooded manakin, Pipra aureola . Morphological and genetic examination of tick specimens led to the identification of six tick species, all belonging to the Amblyomma genus: A. longirostre (n = 230), A. geayi (n = 52), A. varium (n = 4), A. cajennense (n = 2), A. calcaratum (n = 1), and A. humerale (n = 1). A. longirostre was found in 25 bird species, A. geayi in 11, and the four other Amblyomma species in one or two bird species each , mostly located along the coastline (Table ts Table . The 290Amblyomma spp. specimens through high-throughput 16 S rDNA sequencing showed that Borrelia was present in 20 specimens (6.9%), corresponding to 20 larvae. Infection was detected in 12 of the 230 A. longirostre specimens (5.2%) and eight of the 52 A. geayi (15.4%) specimens, but not in A. varium, A. cajennense, A. calcaratum, and A. humerale , the crimson-hooded manakin P. aureola (two sites), the blue-backed manakin C. pareola (one site), the pale-breasted thrush Turdus leucomelas (one site), the silver-beaked tanager Ramphocelus carbo (one site), the straight-billed woodcreeper Dendroplex picus (one site), the black-headed antbird Percnostola rufifrons (one site), and the white-throated Xenops minutus (one site) (Table Borrelia in Amblyomma spp. varied from 0 to 28.5% depending on the sites and sampling dates (Table p > 0.10).Examination of the 290 Borrelia 16 S rDNA (710 bp), flaB (540 bp), gyrB (1172 bp), groEL (600 bp), and glpQ (784 bp) genes further confirmed the presence of Borrelia in the 20 Amblyomma spp. specimens earlier found positive by high-throughput 16 S rDNA sequencing. On the basis of these five gene sequences, no nucleotide variation was observed in the Borrelia from the A. longirostre and A. geayi positive specimens, showing that only one Borrelia species was present. None of the 16 S rDNA, gyrB, groEL, and glpQ gene sequences observed in this study is 100% identical to other Borrelia sequences available on GenBank. However, based on partial flaB gene sequences (323 bp), this Borrelia strain is 100% identical to the Borrelia sp. clone 2 T (GenBank accession number: MN064675) recently found in A. longirostre from Brazil16. A maximum likelihood (ML) analysis based on these flaB gene sequences revealed a robust clustering of the new Borrelia of French Guiana with the Borrelia sp. clone 2 T of Brazil, along with several other Borrelia sp. found in A. maculatum of Texas18, suggesting that all these Borrelia belong to the same species  to infer the exact relatedness of this Borrelia cluster with other Borrelia species and groups  surveys of the equences 23 bp, thBorrelia isolate (A-FGy1 hereafter) after 6 weeks of cultivation from a freshly molted tick specimen, obtained from an engorged A. longirostre larva collected from a wild passerine. The Borrelia A-FGy1 isolate has a typical Spirochaetes helical-shaped structure of ca. 0.2–0.3 μm diameter and 10–25 μm length  in the 16 S rDNA sequence, suggesting that two closely related Borrelia strains are present in the study area. This SNP is upstream the 16 S rDNA V4 hypervariable region we sequenced through high-throughput sequencing, and was thus not detected in our primary bacterial bardocing investigation. However, Sanger sequencing of 16 S rDNA PCR product and Illumina complete genome sequencing consistently show that this SNP is well present in the Borrelia A-FGy1 isolate.We obtained a viable th Movie . The groBorrelia A-FGy1 isolate produced 230,143,188 paired-end reads of 150 bases, which were assembled into 42 contigs with a mean coverage depth of 1,849×. The final Borrelia A-FGy1 genome is 1,236,294 bp in size and consists of a 918,483-bp linear chromosome and 41 contigs of putative plasmids   including 1,055 predicted protein-coding genes, 37 RNA genes, and 31 pseudogenes. The linear chromosome of the Borrelia A-FGy1 isolate had greatest average nucleotide identity (ANI) with members of the third Borrelia group, B. turcica (90.2%) and B. tachyglossi (82.3%).The sequencing of the p) Table . OverallBorrelia species showed that the Borrelia A-FGy1 isolate clusters within a robust clade with the two known species of the third Borrelia group, B. turcica and B. tachyglossi  present in the linear chromosome of 18 other ssi Fig. . The clooup Fig. .Figure 4Borrelia A-FGy1 genome is roughly similar to the genomes of the two other sequenced members of the third Borrelia group, B. turcica and B. tachyglossi. The Borrelia A-FGy1 linear chromosome shows an extensive synteny with chromosomes of other Borrelia species and, compared with LD, RF, and the third group of Borrelia chromosomes, it exhibits a comparable size and organization , B. turcica (lp35), RF species , and with the circular LD plasmid cp26  was partly collinear with the large linear plasmids (megaplasmids) known exclusively from Borrelia species of the third and RF groups. A few plasmid contigs , however, were highly syntenic with LD, but not RF, Borrelia plasmids  did not share any common genetic architecture with any previously known Borrelia plasmids. However, genes located on these plasmids were homologous to either LD or RF Borrelia genes.The ion Fig. . The sinmes Fig. . Likewisp26 Fig. . Only onids Fig. . AnotherBorrelia group members  shared 806 genes while the core genome of the genus Borrelia contained 638 genes, including 590 on the chromosome and 148 on plasmids  that were not present in the LD and RF Borrelia genomes , mostly located on plasmids and not present in other Borrelia genomes  which are inserted within the rRNA operon of B. turcica and B. tachyglossi but that are absent in Borrelia A-FGy1. The Borrelia A-FGy1 chromosome also harbors a conserved RF-like gene architecture of the rRNA operon: there is only one copy each of the 23 S rRNA and 5 S rRNA genes (duplicated in LD Borrelia), and a horizontally acquired set of three purine salvaging pathway genes  that are inserted between the 23 S rRNA and 16 S rRNA genes (present in RF but absent in LD Borrelia species). The Borrelia A-FGy1 genome also contains 19 unique orthologs with B. tachyglossi, B. turcica and all RF Borrelia that are absent in all LD species, including genes involved in important cellular functions such as glycerophospholipid metabolism  and DNA repair   gene and several hypothetical protein genes  of third es Table . Borrelies Table . Some geR) Table . Finallyes Table .Figure 5Borrelia A-FGy1 genome harbors a number of previously described genes encoding immune-reactive Borrelia proteins, including the Flagellin (flaB) and Borrelia membrane protein A (BmpA) genes located on the linear chromosome and an outer membrane protein OspC homolog gene located on plasmid. Three Borrelia plasmid contigs contain variable large protein (vlp) and variable small protein (vsp) genes homologous to those used in the multiphasic antigen variation system of RF Borrelia to evade detection by the host immune system. Three Borrelia A-FGy1 plasmid contigs contained vlp genes: Borrelia p6A-FGy1, p33A-FGy1, and p35A-FGy1, which contained one vlp gene each. One vsp gene was identified on the Borrelia p6A-FGy1 plasmid contig and shared high homology to B. turcica vsp35, B. hermsii vsp24, B. turicatae vspB, and OspC from LD Borrelia species.The Candidatus Borrelia mahuryensis” for this novel bacterium associated with the passerine-associated ticks A. longirostre and A. geayi. The specific name refers to Mount Mahury, French Guiana, which was the first sampling site where we detected the presence of this bacterium.On account of these distinct and coherent microbiological, phylogenetic, and evolutionary traits described above, we propose the designation “Borrelia species, Cand. Borrelia mahuryensis, divergent from the LD and RF Borrelia species, is common in passerine-associated ticks in tropical rainforests of French Guiana. We obtained a pure culture isolate of Cand. Borrelia mahuryensis (A-FGy1 isolate) that is morphologically similar to those of other Borrelia species. This novel Borrelia species is more closely related, although distinct, to the two known species of the third Borrelia group, B. turcica and B. tachyglossi. At the five loci extensively examined, only one SNP was observed between all our Cand. Borrelia mahuryensis DNA samples, showing that at least two very closely related strains are circulating in French Guiana. The recent detection of Borrelia isolates in Brazil17 sharing the same flaB sequence with Cand. Borrelia mahuryensis, along with the presence of closely related strains in Texas18, suggests that Cand. Borrelia mahuryensis may have a broad geographic distribution across American countries, with a substantial intraspecific variation. Finally, examination of the Cand. Borrelia mahuryensis A-FGy1 genome confirmed its difference from other Borrelia species, but revealed the presence of shared features with either LD or RF or both Borrelia species.We found that a novel Cand. Borrelia mahuryensis from 2012 to 2018 confirms that infection persists durably in French Guiana through its circulation in at least two tick species, A. longirostre and A. geayi. Infected tick specimens were collected from eight passerine species, suggesting that these vertebrates can be natural hosts for Cand. Borrelia mahuryensis. Interestingly, the related Borrelia strains detected in Brazil were also detected in A. longirostre16 collected from ground-dwelling birds, and those detected in the United States were detected in A. maculatum18, a species that also parasitizes birds among other hosts23. All of this evidence supports the hypothesis of birds as natural hosts for Cand. Borrelia mahuryensis. However, A. longirostre, A. geayi, and A. maculatum are also known to be associated with a variety of vertebrate species, with the immature stages usually feeding on passerine birds and the adults feeding instead on arboreal mammals such as new world porcupines and sloths (for A. longirostre and A. geayi)25 or large mammals such as cattle (for A. maculatum)23. Natural hosts of Cand. Borrelia mahuryensis can thus be either passerine birds, or mammals, or both. The observation that tick larvae are commonly infected suggests the existence of two non-exclusive scenarios: (i) passerine birds are the hosts, with tick larvae acquiring Borrelia through feeding on infected passerine birds; (ii) mammals are the hosts, with tick females acquiring Borrelia through feeding on infected mammals and further transmitting the infection transovarially to their larvae. Transovarial transmission, however, is not a common feature in the Borrelia genus since it has been demonstrated often for RF, but rarely for LD, species29. Interestingly, the isolation of Cand. Borrelia mahuryensis in culture from a freshly molted tick demonstrates that infection is maintained through molting to subsequent developmental stages in the tick host. Thus, through this transstadial transmission, infected ticks have the potential to infect animals on which they subsequently feed, including humans on whom A. longirostre and A. maculatum can occasionally feed31. Furthermore, while both A. longirostre and A. geayi are native to South and Central America34, migratory birds also regularly introduce A. longirostre to North America39. Bird migration  and cattle transportation (for A. maculatum) may be two important factors affecting the distribution of Cand. Borrelia mahuryensis over long distances and across geographical barriers, thereby explaining its wide geographic distribution.The repeated detection of Cand. Borrelia mahuryensis is more similar to those of the Borrelia third group, B. tachyglossi and B. turcica, which were also recently sequenced19. Members of this group shared similar gene content, including specific genes not present in LD or RF Borrelia. When compared with LD or RF Borrelia species, the genome of Cand. Borrelia mahuryensis shows more similarities with RF Borrelia species and harbors genes not found in LD Borrelia. However, it also harbors LD-specific genes and plasmids, showing that Cand. Borrelia mahuryensis exhibits intermediate features between the RF and LD groups. The Cand. Borrelia mahuryensis genome encodes several immunogenic vlp and vlp proteins (used in the multiphasic antigen variation system of RF Borrelia to evade detection by the host immune system40) as well as a number of immune-reactive proteins, including flab and BmpA, which are used for the diagnosis of LD41. Altogether, these genomic features suggest that Cand. Borrelia mahuryensis and other members of the third Borrelia group form a continuum of Borrelia species between the LD and RF groups, affecting our ability to clearly distinguish between these two groups.The genome of Cand. Borrelia mahuryensis shows that the third Borrelia group is both widespread and biologically diverse. Most of the known members of this group were found in association with reptile hosts21, with the exception of one echidna host12. However, the description of Cand. Borrelia mahuryensis now suggests that members of the third Borrelia group have a large host spectrum that may also include a variety of birds and mammals as natural hosts. It is perhaps not unexpected to find other members of the third Borrelia group infecting a wide diversity of vertebrates. They have RF-like genomes with several housekeeping and macronutrient metabolism genes only present in the RF Borrelia species. Paralogous vlp and vsp proteins playing key roles in the RF multiphasic antigenic variation system and pathogenicity are also conserved, but Cand. Borrelia mahuryensis also have LD-specific orthologs. Future studies should give pivotal clues about the biology of Cand. Borrelia mahuryensis, as recently done through in vitro experiments with other members of the third Borrelia group: Indeed, variable levels of B. turcica resistance to vertebrate serum suggest that tortoises are reservoir host species while birds or humans are not42. Additional studies of Cand. Borrelia mahuryensis are needed to determine its transmission cycle and to establish whether these bacteria are pathogenic for birds and mammals, including humans.To conclude, the description of n = 290). An additional batch of ticks (n = 19) were collected from the Rémire-Montjoly site (site 14B in Table S1) and were taken alive to the laboratory for Borrelia cultivation (see below). All ticks were morphologically identified to species level using morphological and genetic diagnostic criteria25. The global connectivity between tick and bird species was visualized using the network analysis software package Gephi43.Birds were captured using mist nets during the dry season 2012–2018) at 16 sites within forest patches  and universal primers (16 SV4F: 5′-GTGCCAGCMGCCGCGGTAA-3′ and 16SV4R: 5′-GGACTACHVGGGTWTCTAATCC-3′)44 as previously described45. Amplified bacterial 16S rDNA products were purified and sequenced using an Illumina MiSeq platform  and 250-bp end sequence reads were obtained. All bioinformatic analyses were conducted using the pipeline Frogs46 as previously described45. Sequences with 97% similarity were clustered together and identified as an operational taxonomic unit (OTU). Each representative OTU sequence was aligned and taxonomically assigned using the Silva database (https://www.arb-silva.de/).Tick whole-body DNA was extracted using an extraction kit according to the manufacturer’s instructions (Qiagen). The presence of Borrelia species identification were performed through the amplification of flaB, gyrB, groEL, glpQ, and 16 S rRNA gene fragments using specific primers (Table http://www.technelysium.com.au/chromas_lite.html), and alignments were performed using CLUSTALW47, implemented in the MEGA V7 software48.Independent PCR assays for rs Table . All PCRn = 19) were taken alive to the laboratory for blood meal digestion. Ticks were kept in humidified chamber (80–90% relative humidity) until molting . Freshly molted ticks (n = 3) were further surface-sterilized with a bleach solution, rinsed with PBS before being individually cut into two parts. The two parts of the ticks were transferred together into a 6 ml-tube to isolate borreliae that was further cultured at 34 °C in BSK-II modified medium  in anaerobic conditions49 for several weeks and regularly examined under dark-field microscopy. Presence of Borrelia in remnants of the ticks was investigated through a specific PCR assay targeting the flab gene . DNA-seq experiments were performed on an Illumina MiniSeq via a platform  using Illumina MiniSeq Mid Output Reagent Cartridge with a paired-end read length of 2×150 bp. A total of 6 GB of data were obtained. The quality of Illumina reads was analyzed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and reads were further cleaned and trimmed using Trimmomatic50. The remaining reads were assembled into contigs and then into scaffolds with the SPAdes v3.8 assembler51. The annotation was performed via the NCBI prokaryotic genome annotation pipeline52. Dot plot analyses were done using YASS53. Whole-genome alignments were performed using Mauve54.One 55 and roary56. Roary was run with default parameters that are well tailored to infer of the most accurate number of genes56. Paralogs were then split according to the authors methodology. Briefly, if multiple genes from the same sample were found in an orthologs cluster, the neighborhood −5 genes up and downstream - of putative paralogs are used to infer information about synteny and split the cluster accordingly. This can be problematic for mis-assembled contigs or for multigenic family repeated in tandem, but allow genes with similar sequence to be placed in different cluster if they lie in different genomic position.Pangenomic analysis (presence/absence of genes in the 19 complete genomes) was conducted with prokka57 with default parameters was used to remove poorly aligned positions and to obtain unambiguous sequence alignments. Closely related organisms obtained from GenBank were also included in the analyses. The evolutionary models that best fit the sequence data were determined using the Akaike information criterion with the program MEGA v748. Tree-based phylogenetic analyses were performed using maximum likelihood (ML) analyses. ML heuristic searches using a starting tree obtained by neighbor joining were conducted in MEGA v748. Clade robustness was assessed by bootstrap analysis using 1,000 replicates.For analyses of single gene sequences, the GBLOCKS program58. All other complete Borrelia genomes were obtained from GenBank. Multiple orthologs were aligned with MAFFT59 v7. The concatenated multiple alignment was cleaned with trimal. The phylogenetic tree was computed by RaxML v8.2.460 using an ML approach with a GAMMA-LG model and 1,000 bootstrap replicates.A phylogenomic approach was followed using ITEPAll animals were handled in strict accordance with good animal practice as defined by the French code of practice for the care and use of animals for scientific purposes, established by articles R214-87 to R214-137 of the French rural code. All captures were performed by competent people without causing avoidable pain, suffering, distress, or lasting harm to the birds. Bird sampling was done under permission granted by several organizations: the Direction de l’Environnement, de l’Aménagement et du Logement (DEAL) from Guyane, the Direction Régionale de l’Office National des Forêts (ONF) de Guyane, the Conservatoire du Littoral (CEL) de Guyane, the Collectivité Territoriale de Guyane (CTG), the Centre National d’Etudes Spatiales (CNES), the Centre Spatial Guyannais (CSG). The use of the genetic resources was declared to the French Ministry of the Environment (reference TREL1902817S/156), in compliance with the Access and Benefit Sharing procedure implemented by the Loi pour la Reconquête de la Biodiversité.Supplementary information.Movie S1. Microscopy movie of the culture of Cand. Borrelia mahuryensis A-FGy1. Three motile bacteria are apparent in this low-density culture medium.Table S1.Table S3.Table S4.Table S5.


Acute gastroenteritis (AGE) is a highly transmissible condition spreading rapidly between individuals and within households. Rotavirus vaccination was introduced in the UK in 2013. The study objectives were to investigate how acute gastroenteritis incidence changed over 25 years and household incidence of AGE since 2013.Repeated cross-sectional study of Royal College of General Practitioners Research and Surveillance Centre network. We used a negative binomial model to report incidence rate ratio (IRR) using the last 5 years data. We also conducted a retrospective cohort analysis, using a shared gamma frailty model (2013–2017). We explored the impact of child under 5- years, household size, socioeconomic status quintile, and rurality.p < 0.001) compared with households without; the IRR fell across IMD quintiles, for example there is a 37% decrease in incidence comparing IMD quintile 1 to quintile 5 ,In the cross-sectional analysis, the IRR of AGE in households with a child of under 5 years was 12.20 . In addition, we observe a reduction in risk of presentation from the most to the least deprived socioeconomic quintile (second quintile: HR = 0.74 (95%CI 0.59–0.92), to least deprived quintile, HR = 0.55 (95%CI 0.41–0.74). We saw a lower association with male gender, white ethnicity and living outside London, but an increased association with increasing household size.The cohort study revealed that the presence of an under 5 in the household was associated with a higher risk of household presentation (HR = 6.29, 95% CI 5.61–7.06, The incidence of AGE has changed over time: pre-school children, larger households, and living in London were associated with higher rates, and male gender and higher economic status associated with lower rates. Acute gastroenteritis (AGE) contributes significantly to the burden of infectious diseases as well as having wider societal impact . AGE is Children under 5- years are the most vulnerable group to develop AGE which is usually associated with rotavirus infection. Because rotavirus is the most prevalent cause, the World Health Organisation (WHO) recommends rotavirus vaccination for this age group . DespiteWhilst it is known that children aged under 5- years are important carriers of AGE , 8 and aWe carried out this study to describe household incidence of medically attended AGE. We report change in AGE incidence over the last 25 years, and investigated whether we could detect any change in household incidence in the last 5 years, since the introduction of rotavirus vaccine. We wished to describe, and if so to what extent the presence of children under 5 years old in a household are associated with a higher incidence of AGE.We have published a detailed protocol for this study, it has two parts: a 25-year repeated cross-sectional study and a five-year retrospective studies of household incidence .We used the Royal College of General Practitioners (RCGP) Research and Surveillance Centre (RSC) database, a well-established sentinel network database consisting of pseudonymised health care records of individuals in England to conduct this study . The UK We carried out a yearly cross-sectional analysis repeated over 25 years (1st January 1992 until 31st December 2017), reporting incidence over this period and the characteristics of this population. We also conducted two five-year studies of household incidence; we explored the incidence in the last 5 years in cross-sectional data and also conducted a retrospective cohort study (1st January 2012 to 31st December 2017). We restricted our exploration of household incidence to the last 5 years since the introduction of rotavirus vaccine in the UK  we also,We used descriptive statistics to report any change in the population with AGE over the last 25 years, with stratification by age, gender, ethnicity and obesity, measured using body mass index (BMI). We used the World Health Organisation (WHO) classification of obesity in adults: Underweight (BMI < 18.5), normal (BMI 18.5–25), overweight (BMI 25–29.9), class 1 obesity (BMI 30–34.9), class 2 (BMI 35–39.9) and finally class 3 (BMI ≥40) . All of n = 4346 for the cross-sectional study, and n = 3967 for the retrospective cohort) over the previous 5 years. We directly standardised AGE rates by age and gender, using the 2011 national census population [We defined a case of household incidence when two members of the same household presented on the same day or within 10 days. We identified cases of household incidence  hence our adoption of the negative binomial model. We controlled for deprivation using the index of multiple deprivation (IMD) quintile, the urban-rural status of the household, their NHS Region and the cross-sectional year. We used Office of National Statistics (ONS) data, linked to post code, to group practices into conurbation, city and town, or rural; this is based on population density. We also looked for any north-south difference using English NHS Regions. These divide England into: north, midlands and east, south, and London. We fitted the negative binomial model using the MASS library in R, version 7.3–45 and report incidence rate ratios (IRRs) with 95% -confidence intervals.We employed a negative binomial model to study the potential impact of the presence of a child under 5 years old in a household on the incidence of gastro-enteritis. Data consisting of counts of transmissions of AGE in households was tested for over-dispersion , IMD quintile , Urban-Rural classification , Ethnicity, a 5 level categorical variable consisting of Asian (reference), Black, Mixed, Other and White. NHS Region  because descriptive data suggested there was no difference in incidence. Our exploration dividing children into those under 2 years is included in the supporting information files  Table.After an early steady rise in both crude and standardised rates we observed a rise in the rate from 1992 through to the middle of the first decade of the new century then a major decline towards the end of our observation period. The later years appear to be clearly differentiated from the early years , compared with 919/100,000 registered (95%CI 902–935), respectively Fig.  Tables. 2) who presented more. Underweight adults had the highest rate of gastroenteritis, though it fluctuated over time. , compared to a household without an under 5-years old resident. Compared to the most deprived quintile other quintiles show a reduction in incidence . Compared with the reference category City and town (medium density suburban housing), conurbations (highest population density) showed a decrease in incidence by 24%. A rural setting did not show any difference. All the other NHS Regions had lower rates of household incidence, around half that of London (p < 0.0001). The observed fall per year in incidence rates was not statistically significant.In the 5 years data 2013–2017), there were 4346 incidence cases. We report the IRR from our negative binomial model . Approximately 27% of households with two or more people had a child under 5 years (though this varies from year to year). The median household size was 3.0 . We also included gender, ethnicity, IMD urban-rural classification, and NHS Region in our model . There is a decreasing trend in hazard ratios for IMD quintiles from most deprived category to least deprived: 0.74 (95%CI 0.59–0.92) for the second more deprived quintile through to 0.55 (95%CI 0.41–0.74) for the least deprived quintile. Overall, male gender was associated with less AGE . White ethnicity  had a lower HR than Asian but there were no other statistically significant differences. Each additional member of a household increased the HR of household incidence . Conurbations had less household incidence than city and town, but there were no differences in rural settings. London NHS Region had a greater hazard of AGE than other parts of the country  because our system of household linkage was not in place prior to this, though this conveniently fitted with the period after the introduction of rota virus vaccine. If we could have assumed that households had not changed in the previous 20 years, we could have identified more cases . We felt it was unsafe to include these in our analysis. Whilst this is a limitation, it also demonstrates that household incidence continues notwithstanding the introduction of rotavirus vaccine [We only reported the cases of household incidence in the last 5 years , it is possible that the propensity to consult and the threshold for presentation may have changed over this period . We onlyThis study suggests it is feasible to detect household incidence in a sentinel network, using routine primary care data. Household incidence of other infections should also be quantified, particularly influenza where the introduction of childhood vaccine aims to target the carriers the disease.Research is also needed to explain why underweight adults more frequently present with AGE. They should possibly be tested for malabsorption and/or intolerances.Household incidence of gastroenteritis can be detected from routine data collected within a sentinel network. Household incidence of AGE continues, notwithstanding the introduction of rotavirus vaccine. Pre-school children, larger households, city and town location (medium density housing), and London were associated with higher risk. Male gender, higher economic status, and conurbations outside London were associated with lower rates. Further public health interventions are required to reduce the spread and incidence of AGE.Additional file 1: Figure S1. Incidence of gastroenteritis  in RCGP RSC sentinel practices 1992-2017 (standardised against the 2011 Census).Additional file 2: Figure S2. Crude gastroenteritis rates age band 05-17years.Additional file 3: Figure S3. Crude gastroenteritis rates age band 18-64years.Additional file 4: Figure S4. Crude gastroenteritis rates age band 65 and above.Additional file 5: Figure S5. Crude gastroenteritis rates age band 00- 4years.Additional file 6: Figure S6. Gastro incidence rates per 1000 total by age bands.Additional file 7: Figure S7. Crude gastroenteritis rates by ethnicity.Additional file 8: Figure S8. Household incidence gastroenteritis ethnicity rates.Additional file 9: Table S1. 2011 census data and proportion.Additional file 10: Table S2. Age and sex standardised rates.Additional file 11: Table S3. Gastroenteritis rates.Additional file 12: Table S4. Gastroenteritis rates with confidence interval.Additional file 13: Table S5. Title: Gastroenteritis rates age and sex.Additional file 14: Table S6. Crude gastroenteritis rates by ethnicity.Additional file 15: Table S7. Ethnicity.Additional file 16: Table S8. Gastroenteritis rates by age band including under 2 years old.Additional file 17: Table S9. Variance inflationary factors associated with the Frailty Survival Model.Additional file 18: Table S10. Acute gastroenteritis rates over 25 years, showing rates in boys and girls under five years old and males and females five years and over. Table S11. Acute gastroenteritis rates, in adults by combined WHO obesity category. Table S12. Rates of household incidence by age-band. Table S13. Rate of household incidence by socioeconomic status reported in IMD. Table S14. Household incidence of AGE by household size. Table S15. Household incidence of AGE by household type. Table S16. Household incidence of AGE by urban-rural classification.


Although gender-related disparities in cardiovascular surgery have been investigated extensively in the past decades, knowledge about the impact of gender on outcomes after surgery for acute aortic dissection type A (AADA) is sparse. This study investigated the impact of gender on early morbidity and mortality and follow-up outcome in patients after surgery for AADA and to analyze gender-related risk factors for 30-day mortality.This retrospective study included 368 consecutive patients  undergoing surgery for AADA between 2001 and 2016 at our department. Survival was estimated by Kaplan-Meier curves. Risk factors for 30-day mortality were assessed by multivariable logistic regression and interaction analysis.p <  0.001) and showed a higher logistic EuroSCORE I . In the male group, a higher portion of smokers  and intraoperatively, more complex procedures and longer cardiopulmonary bypass (CPB)  and cross-clamping times  occurred. 30-day mortality was 19.0% in the female and 16.5% in the male group (p = 0.545). Predictive for 30-day mortality in both genders was intraoperative blood transfusion, while in the female group chronic obstructive pulmonary disease (COPD), peripheral arterial disease and preoperative intubation were predictive. Preoperative cardiopulmonary resuscitation and duration of CPB time were predictors only in males. Averaged follow-up time was 5.2 years and survival did not differ between genders, even if it was stratified by age over 70 years.Women were older (70.7 years vs. 60.6 years; This analysis demonstrated a similar and satisfactory survival in both genders after surgical treatment of AADA. Women and men differed significantly in age, unadjusted and adjusted risk factors and complexity of surgical treatment, but gender itself was no risk factor for mortality. These results suggest that the decision-making for surgical treatment should not depend on gender, but that accounting for sex-specific risk factors rather than common risk factors may help to improve the outcome in both genders. Acute aortic dissection type A (AADA) is one of the major causes of death in cardiovascular disease for women and men , 2 and rStudies on gender-related differences after surgical repair of AADA are rarer and outcomes are controversial. A higher morbidity and mortality in women compared to that of men was reported previously , 5, 11. The aim of this large retrospective observational study was to investigate the differences between women and men operated on for AADA regarding clinical presentation, management, surgical technique and outcomes in terms of morbidity, complications and survival. Gender-specific risk factors for 30-day mortality were detected based on a comprehensive database.From January 2001 to November 2016, a total of 368 consecutive patients suffering from AADA, 126 women and 242 men underwent surgical treatment of AADA at our department. AADA was defined as any dissection of the aortic wall with a clear entry site or intramural hematoma involving the ascending aorta or with an entry in the descending aorta with retrograde dissection. The diagnostic tool in all cases was computed tomography; echocardiography was used as confirmation when possible. Angiography and magnetic resonance imaging were not performed routinely. Primary endpoint was 30-day-mortality; secondary endpoints were peri- and postoperative complications and follow-up survival.Pre, intra- and postoperative variables were extracted retrospectively from electronic patient charts. They included demographics, comorbidities, history, clinical presentation, imaging findings, management, peri- and postoperative events and mortality. Predicted 30-day-mortality was calculated by logistic EuroSCORE I and EuroSCORE II  , 16. Fol2 insufflation was used in every case. After insertion of the perfusion cannula directly in the graft CPB restarted again. The proximal anastomosis and any required extra procedures (coronary artery bypass grafting (CABG), valve, etc.) were performed during re-warming.A standard median sternotomy followed by longitudinal pericardiotomy was performed under general anesthesia. All operations were performed by senior surgeons. The cardiopulmonary bypass (CPB) was performed with moderate hypothermic cardiac arrest (MHCA) with a nasopharyngeal temperature between 20 and 24 °C. In the early years either echo guided direct cannulation of the distal ascending aorta or, after surgical cut-down, the femoral artery was used for arterial cannulation in the most of cases. Since 2010, a transatrial cannulation of the left ventricle via the right upper pulmonary vein was used as standard technique for arterial cannulation . Venous t-test. Not normally distributed data as well as ordinal data were shown as median and the 25th and 75th percentiles or, where appropriate, as range and compared by Mann–Whitney U-test. Categorical data were summarized as absolute (n) and relative (%) frequencies and compared by Chi2-test or Fisher’s exact test. Missing data were excluded pairwise. Variables with missing data > 5% are marked in the results tables.Characteristics of women and men were compared by univariate statistics. Normally distributed continuous variables were presented as mean ± standard deviation and compared by unpaired p-value of 0.1 or less  and elderly patients (≥ 70 years) and was compared regarding gender-specific differences by log-rank test.p-value of ≤0.05 was considered statistically significant. Data were analyzed with IBM SPSS Statistics for Windows (Version 24.0).All tests were conducted 2-sided and a 2 vs. 2.1 m2) and a lower body mass index (BMI 25.7 kg/m2 vs. 26.8 kg/m2) compared to men, but the percentage of obese (BMI > 30 kg/m2) patients was similar (18.4% vs. 21.1%). Men were more often smokers than women (27.6% vs. 16.0%). Male patients had significantly higher creatinine levels than female patients (98.1 μmol/l vs. 80.0 μmol/l), but the percentage of patients diagnosed with renal insufficiency or failure did not differ statistically between genders. According to the classification by DeBakey, women and men tended to present with a different extent of aortic dissection, however this difference was not statistically significant (p = 0.053). There were no further significant differences with regard to clinical presentations between genders.One hundred twenty-six female patients (34.2%) and 242 male patients (65.8%) were included in the present study Table . Women wp = 0.164) with a larger size of valve prostheses compared to that in women (25 mm vs. 23 mm). Women received more packed red blood cells (4 units vs. 2 units) and hemofiltration (34% vs. 23%) compared to men.Intraoperatively, males underwent more complex procedures when operated on for AADA and accordingly longer procedural times were found compared to those in the female group. Women received isolated supracoronary ascending aortic replacement (61.9% vs. 44.6%) more often while men received more David operations (8.3% vs. 1.6%) and other complex procedures, but the latter without reaching statistical significance Table . In consPostoperative complications were more frequent in the male group. Blood drainage loss within 48 h postoperative was higher in men and likewise the total number of red blood cells, fresh frozen plasma and of platelets (see Table n = 339). Median follow up time was 5.2 (1.9–8.4) years, and did not differ between genders (p = 0.542). Kaplan-Meier analyses showed no significant differences in all-cause survival between women and men (p = 0.575). Median estimated survival time was 8.9 (5.9–11.8) years in the female group and 9.5 (6.1–13.0) years in the male group. Cumulative survival of women and men was 77 and 76% after 1 year and 48 and 50% after 10 years . Detailed information on variables associated with 30-day mortality are provided as Crude predictors for 30-day-mortality differed substantially between the overall group, the female and the male group Table . These fIn the multivariable analysis on pre- and intraoperative variables adjusted overall predictors for 30-day mortality were CPB time, number of intraoperatively administered red blood cell units, additional CABG and particularly cardiopulmonary resuscitation within 48 h preoperative Table . In the In accordance with the interaction analysis, the multivariable analysis, in which only preoperative variables were included Table  revealedOur large study compared early and late outcomes between women and men following surgical repair of AADA and assessed gender specific risk factors for 30-day mortality. Our main findings are that women were about 10 years older at the time of surgery and that men underwent more complex and longer surgery times with concomitant more complicated postoperative course. Early and late mortality did not differ significantly, though women and men showed substantial differences regarding unadjusted and adjusted risk factors for 30-day mortality.Nienaber et al.  presenteSeveral studies indicated a poor outcome after surgery for AADA in women , 11 and Age is a risk factor for early mortality in AADA in several studies , 21, espMen and women presented with only few differences in preoperative findings, despite for age and EuroSCORE. As described previously , smokingIn contrast to previous findings women did not present with a higher incidence of arterial hypertension in our study despite their higher age , 5, 11. Though men were younger and did not present with a considerably worse preoperative state, postoperative complications were more frequent compared to those in women, presumably associated with the higher portion of complex surgery. The decision to perform less complex surgery might be related to the older age of female patients and the usually less elaborate surgical treatment that is recommended in elderly patients to shorten bypass-time , 26. AccIntraoperative blood transfusion was a risk factor for 30-day mortality in both genders, but despite shorter procedural times and less complex surgery, female patients received more red blood cell units intraoperatively compared to male patients. Though data is lacking in our analysis, many studies showed that women have lower preoperative hematocrit prior to cardiovascular surgery, a higher rate of red blood cell transfusion and greater hemodilution on cardiopulmonary bypass compared to men , 28. In Despite the observed gender-specific differences, women and men had an acceptable 30-day mortality of 19.0% vs. 16.5% and a total 30-day mortality of 17.4%, overall comparable to the results of Boening et al. and Rylski et al. who reported a total 30-day mortality of 16.9% , 16.6% i. Solely in the female group, PAD, coronary heart disease, previous PCI, previous cardiac surgery, as well as COPD, hyperlipoproteinaemia and chronic renal insufficiency were identified as specific predictors, which are predominantly associated with known age-related comorbidities atherosclerosis and prior cardiac surgery [Crude and adjusted risk factors for 30-day mortality showed major differences between genders surgery . Moreove surgery . In cont surgery , howeverIn the female group COPD was identified as strong predictor for 30-day mortality. COPD was a risk factor for in-hospital mortality after aortic surgery in a large analysis from the Japan Adult Cardiovascular Database . AlthougPatients with coronary malperfusion who underwent additional CABG with surgery for AADA showed a higher unadjusted risk for 30-day mortality in all groups. Malperfusion of different organs affected by dissection of the aortic branch vessels is one of the main complications of AADA and is a known risk factor for early mortality after surgical repair of AADA . HoweverThough the clinical presentation of women and men was comparable, preoperative cardiopulmonary resuscitation as strong risk factor for a poor outcome  showed aWe therefore conclude that in general it is important to include intraoperative factors in multivariable analysis in order to draw conclusions about all risk factors for early mortality. However, a detailed separate analysis of the preoperative risk profile may be useful to predict the mortality in patients prior to surgery for AADA.Kaplan-Meier analyses showed no significant differences in survival between women and men in our study as in recent studies , 12, 23.In summary, women and men showed a comparable short- and long-term survival after surgery for AADA, though women were significantly older and presented with a different pattern of risk factors and men underwent more complex surgery and had a more complicated postoperative course Fig. . A similThe principal limitation of this single-center study is its retrospective design and thus the issue of missing and incomplete data. The limited sample size in the female patient group and the large confidence intervals of some predictors should be considered. Studies on larger patient populations should further investigate sex-specific risk factors.A major finding of this study is that unadjusted and adjusted predictors for 30-day-mortality differed substantially between the overall patients, women and men. Survival was comparable and satisfactory in both genders and gender itself was no risk factor for mortality after surgical repair of AADA. Consideration of sex-specific risk factors instead of common risk factors may help to improve outcomes in women and men after surgical repair of AADA.Additional file 1: Supplementary Table 1. Pre- and intraoperative variables with univariate association to 30-d-mortality , Supplement to Table


Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins , as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho = 0.5–0.9) with results from respective ELISA. Five proteins  were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p < 0.0001) compared to individual markers. Collectively, our study describes a multiplex assay for absolute quantification and verification analysis of previously described putative CKD prognostic markers, laying the groundwork for further use in prospective validation studies. Disease management is characterized by excessive financial costs 3. Common risk factors for CKD include ageing of the population and increased rates of diabetes and hypertension4. Of note, patients with CKD have an overall 30-fold increased risk for suffering from Cardio Vascular Disease (CVD) complications, this being the main cause of CKD-associated deaths. Specifically, ~45% of patients with CKD stage 4–5 die from CVD5 and risk of CVD increases with CKD severity, which is already significantly higher in early CKD compared to non-CKD7. Early identification of CKD and addressing modifiable risk factors is recommended, as it can reduce the risk of kidney failure and CVD by up to 50%8. Early detection or prediction of complications enable early intervention, thus could increase the chances for higher treatment efficacy11.Chronic kidney disease (CKD), defined as reduced kidney function and/or evidence of kidney damage, is a major public health problem throughout the world. Major health problems around the globe, with consistent prevalence rates of 10–13% have been reported (depending on reference group and stage)13. However, these markers have substantial limitations in evaluating CKD progression. Albuminuria quantification and categorization into normalbuminuria (<30 mg/24 hours), microalbuminuria (30–300 mg/24 hours) and macroalbuminuria (>300 mg/24 hours)14 is commonly used alone or in combination with eGFR to predict kidney damage15. However, a significant proportion of patients progress without exhibiting significantly increased urinary albumin levels16. Collectively, there is ample room for improvement especially with respect to prognosis of CKD complications, in part due to the fact that the main biomarkers currently used  are neither an early indicator nor linked to molecular pathophysiology involved in disease progression, but rather the functional consequences of the already settled pathological modifications.CKD diagnosis is currently based on the detection of reduced estimated glomerular filtration rate (eGFR) and/or albuminuria, as indicators of renal dysfunction11. As such, biomarker research in the field has been rigorous with ample biomarker data collected until now20. Various plasma proteins have been associated with CKD progression, with CVD, and with patient-relevant outcome . These include beta-2 microglobulin (B2M), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1, liver-type fatty acid binding protein, cystatin-C, FGF23, as prominent examples25.More accurate evaluation of progression risk could be of significant benefit, since treatment options are presently available28 and received a letter of support by the US Food and Drug Administration  for use in early detection of nephropathy in diabetic patients29. CKD273 has been found to predict progression at early CKD stages (eGFR > 70 mL/min/1.73 m2) more accurately than albuminuria30, and has been applied in the proteome-guided intervention trial, PRIORITY31.Even though associations with outcome have been reported, the overall suboptimal accuracies of individual markers emphasize the need to establish panels or multi-parametric classifiers, better reflective of the substantial phenotypic and molecular heterogeneity of CKD. Along these lines, a urinary peptide classifier, CKD273, consisting of 273 peptides, fragments of multiple kidney-specific as well as plasma proteins detected by the use of capillary electrophoresis (CE) in combination to mass spectrometry (MS) has been developed, subsequently evaluated26. As a step in this direction, we aimed at establishing absolute quantification assays and performing an initial evaluation of the prognostic value of six plasma proteins, when measured in a multiplex format, for a patient-relevant endpoint, death. The employed method is the targeted mass spectrometry-based multiple reaction monitoring (MRM), relying on the quantification of one or more unique/proteotypic peptides corresponding to a target protein33 without using antibodies33. The applied quantification strategy is the Absolute Quantification (AQUA) workflow34, involving stable isotope-labeled peptides, spiked into the sample of interest at predefined amounts and measured simultaneously with the respective endogenous peptides36. The selection of the specific markers was guided by the existence of mass spectrometry-based data for these proteins37, their expected abundance levels based on the existent literature, targeting to avoid extensive pre-fractionation, as well as levels of evidence for the association of these proteins with unfavorable outcome. Using the study by Glorieux et al.37 as a basis, involving high resolution LC-MS/MS analysis of plasma proteome from patients with CKD and reporting on associations to outcome, in combination to the aforementioned criteria, the tested panel includes B2M46 and SERPINF150, having been largely studied in CKD and serving as positive controls for the approach, as well as AMBP, LYZ, HBB, and IGHA1 with some earlier reported associations37, nevertheless not been validated yet in association with disease progression via absolute quantification.Prediction of progression to ESRD or CVD-related endpoints is critical for disease managementet al.37, further considering availability of proteotypic peptides for MS quantification assays, as described in Methods. To avoid extensive pre-fractionation steps potentially compromising assay reproducibility and applicability, only markers with expected relatively high (>100 ng/ml) plasma abundance levels reported in the literature and existing proteomic databases were considered (Supplementary Table S1). The selected marker candidates were B2M, SERPINF1, AMBP, LYZ, HBB, and IGHA1. Following a development phase, involving testing multiple peptides per marker, as described in Methods, standard curves per selected peptide were generated to ensure reproducibility, minimal or lack of matrix effects and linearity in measurements in the range of expected quantified values. Standard curves using synthetic isotope labeled peptide standards (SIS), specific per marker, were generated using a pooled plasma sample , as matrix.The markers to be quantified were selected from the list of differentially expressed proteins in plasma of haemodialysis (HD) patients with CKD stage 5 versus CKD stages 2–3 in Glorieux 2 coefficient of at least 0.992 and for a minimum 4-point concentration ranges, spanning the expected abundances were observed. In addition, CVs of the ratio of the abundance of the standard (SIS) to the endogenous  peptide for all dilution points ranged from 0.40–16.06%, (only exception was for the concentration 72.18 ng/mL of LYZ peptide with CV of 20.79%) suggesting good reproducibility and minimal matrix effects for the assays. The LLOQ (Lower Limit of Quantification) ranged from 9.38–167.5 ng/mL, defined as the lowest qualified concentration level of SIS peptide corresponding to SIS/NAT ratio with CV < 20%53 . Consequently, the LLOD (Lower Limit of Detection) for the peptides was estimated at 6.54–112.52 ng/mL, based on the following formula 3.3*sd/slope: (https://www.ich.org/). (Supplementary Table S2).As shown in Fig. Using the established assays, quantification of the selected markers was performed on all baseline samples from 72 patients that were included in the original study. These included patients with CKD stage 5 (n = 33), CKD stage 4 n = 21), CKD stage 3 (n = 12), and CKD stage 2 (n = 6) at baseline. Thirty-five events (deaths) were observed (mean follow-up time 3.61 ± 2.96 years), with n = 34 patients being alive at the last contact date (mean follow-up time 5.21 ± 2.77 years). For 3 patients, no follow-up information was available. The main clinical data are summarized in Table , CKD staUsing the established assays, high quality spectra were obtained for all but one sample, where the data for SERPINF1, AMBP and B2M did not pass quality control  and advanced CKD (stage 5) with HD, we performed the same comparison with our MRM data  versus combined stages 2–3, except for IGHA1 and SERPINF1, where no significant changes could be detected  Fig. . For thi. ELISA measurements per sample are presented in Supplementary Table To further validate the MRM measurements, ELISAs were performed for 5 out of the 6 proteins where specific and well characterized assays were available . Indeed, the correlation of the findings of the 2 methods was to the least moderate  or in most cases high , Kaplan Meier analysis was performed. Given its overall poor associations to staging as well as lack of correlation to eGFR, IGHA1 was excluded from further analysis. As shown in Fig. To assess the added value of combining the measured markers into a single simple classifier we utilized instance-based learning. In that, a k-nearest neighbor (knn) classifier was fed with the MRM data and was trained to distinguish between disease status at 7 years follow up time. The model was validated in predicting risk for mortality by leave one out crossvalidation. As the levels of SERPINF1 were found to be highly affected by HD Fig. , the mar2  have been described in association with the disease58 but generally are not routinely implemented, yet.Multiple biomarkers for CKD progression towards ESRD and death, widely classified as biomarkers of kidney function and kidney damage, have been described59. A prominent example is the multi-peptide urinary classifier CKD273 being used for the detection of nephropathy at early stages29.To address disease complexity, multi-parametric, high dimensional classifiers incorporating numerous disease-specific and systemic molecular changes are increasingly being established, as better reflecting the disease molecular heterogeneity33. MRM has been widely applied in plasma for proteomic biomarker validation60 and for various plasma proteins (such as apolipoproteins). It has been demonstrated to provide assay performance equal to well characterized ELISA assays61. In fact, the observed correlations between our MRM data and respective independent immunoassays (Rho = 0.53–0.88) are in line to existing literature and reported acceptable correlations of such assays63.We employed a multiplex MRM mass spectrometry assay for the quantification of six previously described, exploratory CKD markers. MRM, in contrast to the classically employed highly sensitive ELISA assays, does not require the use of specific antibodies, thus facilitating multiplexing and increasing specificity via eliminating antibody cross-reactivity problems64. Applications involving combination of MRM with immunoprecipitation protocols62 or peptide separation by two dimensional liquid chromatography can extent the quantification range from below 100 ng/mL to about 500 pg/mL range65. However, these protocols are substantially more elaborate, and not well suited for routine implementation.Besides evidence of association with CKD, the selection of the specific markers was driven by the existence of mass spectrometry-based data per protein to ensure detectability, as well as expected abundance levels to eliminate the need for extensive pre-fractionation. The detection limits observed in our study (in the range of ng/mL) are similar to ones regularly reported when using unfractionated plasma66. It is a component of the class I major histocompatibility complex (MHC), synthesized normally by lymphocytes, filtered in the glomerulus and catabolized by the proximal tubular cells68. Upon defective renal function, its serum levels increase significantly68. In line with our study, gradual increase in B2M levels across stages of CKD has been shown38. Furthermore, several associations between B2M levels and death, dialysis or cardiovascular events have been reported43, in agreement with our findings.Among the analyzed proteins, B2M is the most well-characterized CKD-associated marker and known uremic solute according to the European Uremic Toxin Work Group69, was later defined as a member of the serine protease inhibitor family with antiangiogenic, antioxidative, anti-inflammatory, and antitumorigenic activity70, and was recently assigned a renoprotective role71. In line with our analysis, SERPINF1 serum levels were found elevated in ESRD patients compared to healthy controls47. Similarly, SERPINF1 was reported increased in plasma of CKD3 patients in comparison to controls48. In addition, Hui et al., demonstrated that SERPINF1 levels correlated with eGFR (n = 1136), and further associations to CKD progression  were supported49. In our study, surprisingly, an association of lower SERPINF1 levels with death was detected. This finding is most likely driven by the HD patients included in the cohort. In fact, if we exclude HD patients from the analysis, higher levels of the protein in CKD5 vs CKD4 can be observed , initially characterized as a neuronal differentiation factor in retinoblastoma cellsved Fig. .72 and tubular dysfunction in diabetic nephropathy73. In addition, several shotgun proteomic studies followed by verification via different assays , or ELISA), supported an upregulation of AMPB in patients of various CKD stages compared to healthy controls, in adipose tissue, urine as well as urine exosomes76. AMBP was also found to progressively increase in plasma of individuals with increasing CKD stage –from CKD1-2 to CKD3-4 and finally to CKD5- based on relative quantification by LC-MS/MS78 or MRM77.AMBP has been studied mainly in urine and to a lesser extent in plasma and tissue in the context of CKD. Increased urinary AMBP levels have been associated with kidney damage79. This latter study also revealed elevated levels of these proteins in ESRD patients and recipients with poor graft function compared to patients with good graft function, a finding which was further confirmed by Western Blot analysis for SERPINF1 and AMBP79. Our study also supports the progressive up-regulation of AMPB in plasma with increasing CKD stage, negative correlation to eGFR and unfavorable outcome (p = 0.00054).AMBP, SERPINF1, B2M, and HBB have been investigated as HDL (High-density lipoprotein) associated proteins by LC-MS/MS, and were found to correlate with severe kidney damageet al.37. This finding could not be verified in our study, which also failed to demonstrate association of the protein to eGFR. In contrast, the differential abundance of HBB, one of the two polypeptide chains that form Hemoglobin A80, in CKD5 versus earlier stages was verified and a modest association to eGFR was observed. The most pronounced finding of our study is the clear associations of LYZ with all CKD stages, eGFR as well as survival. LYZ is released from leukocytes and macrophages, and has antibacterial properties and an attributed role in myocardial depression and vasodilation81. With the exception of the study by Glorieux et al.37, and an in vitro study suggesting anti-inflammatory properties of LYZ on human proximal tubular epithelial cells (HK-2 cells)82, studies of LYZ in the context of CKD are lacking. Based on our results, such studies seem well justified, which may also expand to the investigation of cardiorenal syndrome, considering reports associating plasma levels of LYZ to coronary artery disease severity83.In comparison to B2M, SERPINF1 and AMBP, the published evidence associating IGHA1, HBB and LYZ to CKD is limited. IGHA1 was found at higher levels in the plasma of patients with CKD5 with HD in comparison to CKD2-3 in the study by Glorieux ) suggests that our targets could be specific markers for CKD progression. However, the power of the study is too small and further validation is needed to confirm this observation. Overall, the small sample size and restriction to 6 markers are clear limitations. Furthermore, larger scale studies will be required to validate changes per stage including in early disease. The small sample size does not also allow multivariate analysis to be conducted with confidence. An effort to investigate the added value of the classifier on top of clinical variables was performed and even though an independent prognostic value over age could be seen, this was lost with the addition of CKD stage . All methods were performed in accordance with the relevant guidelines and regulations. All individuals gave written informed consent. Pertinent clinical data are provided in Supplementary Table 84.The study population consisted of all 72 patients  that were recruited for the “Urosysteomics” studyhttp://www.nist.gov/] on 05062014] was imported into Skyline software. The Skyline indicated proteotypic peptides and respective transitions per peptide which were further validated with additional bioinformatics tools: The PeptidePicker (http://mrmpeptidepicker.proteincentre.com/peptidepicker9/) that predicts proteotypic peptides with similar criteria as Skyline85; The Peptide Tracker (http://tracker.proteincentre.com/) that records transitions and chromatographic properties of specific proteotypic peptides based on previously performed MRM assays86, and the CPTAC Assay Portal (https://assays.cancer.gov/available_assays) that compiles information of existing MRM assays 87. In addition, shortlisted peptides were evaluated using the Protein Basic Local Alignment Search Tool  to ensure their proteotypicity.For the establishment of the MRM assays, proteotypic peptides were selected considering the following criteria: (i) the peptides uniquely represented the target protein, (ii) lysine or arginine was allowed only at the carboxy terminus , (iii) peptide sequence included 6–25 amino acid residues to ensure acceptable ionization and gas-phase fragmentation, and (iv) amino acids that are susceptible to chemical modifications such as cysteine, methionine should be absent. To perform the selection, multiple tools were employed in combination: a spectral library from the National Institute of Standards and Technology (NIST)  . As the employed labeled peptides were not of highest purity, their purity and final concentrations were defined by MS analysis , parallel reaction monitoring (PRM) in comparison to standard/recombinant protein for B2M, or estimation of the labeled peptide amount by correlating 4 different analytical runs  to respective ELISAs for LYZ.For the peptides identified (4–7 per biomarker) and their respective transitions (3–5 per peptide), based on the aforementioned procedure and considered for further analysis, initial MRM experiments were conducted using two plasma samples  in triplicate. Further shortlisting was performed based on: (i) the quality of the MS/MS spectrum of each peptide in the human spectral library, (ii) score and number of observations in MS-based proteomics experiments, as provided from PeptideAtlas , reduction (10 mM dithioerythritol) and alkylation (50 mM iodoacetamide) the samples were digested with trypsin [(1:100 enzyme: protein ratio (w/w)] for 16 hours in the dark (RT). The peptide mixture was desalted with solid phase extraction zip-tips (Thermo Scientific) and the extracted peptides were dried using a vacuum centrifuge. The dried peptides were solubilized in mobile phase A , pH 3.5 to obtain a final concentration of 0.5 μg/μL. A mixture of the SIS peptides was then added in each sample after drying the peptides as follows: IGHA1: 8000 ng/mL, B2M: 800 ng/mL, HBB: 770 ng/mL, AMBP: 2240 ng/mL, LYZ: 800 ng/mL, SERPINF1: 668 ng/mL. Liquid chromatography was performed using an Eksigent nano-HPLC system, coupled with a C18 analytical column  (Thermo Scientific). Peptide separation and elution were performed with a 60 min gradient of 5–90% mobile phase B  at a flow rate of 300 nL/min. Samples were injected into the LC system and loaded on the C18 column. Tryptic peptides were analyzed on an AB Sciex 4000QTRAP with a nanoelectrospray ionization source controlled by Analyst 1.5 software (AB Sciex). The mass spectrometer was operated in MRM mode, with the first (Q1) and third quadrupole (Q3) at 0.7 unit mass resolution. Detailed information about the acquisition method and the used parameters are provided in Supplementary Table Equal volume (2 μL) of plasma samples containing approximately 100 To define the range and ensure linearity in measurements, a calibration curve for each peptide was generated spiking SIS peptide at different amounts in a CKD pooled plasma sample , in order to maintain the same background matrix to the analyzed samples. Dilution points were selected to cover reported concentration ranges per marker based on the literature and/or reported MS data and each dilution point was analyzed in triplicate. Samples were run in LC-MRM-MS, as described above.91 and all chromatograms were manually inspected to ensure the good quality and accurate peak picking. In particular, manual inspection ensured that the extracted ion chromatograms of the stable isotope-labeled standard (SIS) and endogenous  peptides chromatographically co-eluted and exhibited identical peak symmetry, shape, and width. The top signal producing transition was selected as the quantifier transition in all cases, while the remaining transitions were used as qualifier transitions, for accurate peak profile and retention time confirmation. In the case of standard curve, linear regression analysis was performed to define the slope and linearity of measurements.Data analysis was performed using the Skyline softwarehttps://www.uniprot.org).Finally, the NAT/SIS ratio [Light/Heavy-relative response (RR)] of the quantifier transition was used for quantification (peak area of quantifier transition in NAT peptide/peak area of quantifier transition in SIS peptide). Concentrations were reported to ng/mL by using the protein molecular weights, as determined by Uniprot (The same cohort of samples (N = 72) was analyzed with ELISA to investigate the correlation among the two different methods. The ELISA kits that were employed, specifically, included: for protein bikunin/AMBP: DY7744-05/R and D systems;, for SERPINF1: ab213815/Abcam, LYZ: ab108880/Abcam, IGHA1: ab196263/Abcam, and B2M: ab108885/Abcam. Values could not be obtained for n = 1 samples for SERPINF1 and n = 3 samples for B2M.www.graphpad.com), whereas statistical and survival analysis were conducted in the R (version 3.6.1) environment for Windows utilizing base functions from the packages stats, survival and survminer. Significant changes at the levels of continuous variables across groups were determined with the non-parametric Kruskal-Wallis and Mann-Whitney tests. Kaplan-Meier plots were constructed to compare survival between Low and High protein concentration (ng/mL) groups, defined by a median cut-off point per protein, across samples. Survival curves were compared for significant differences (p < 0.05), from baseline diagnosis to 7 years follow-up time with the Log-rank test. Similarly, correlation analysis between eGFR and MRM quantifications were performed. Linear relationships between the continuous variables were assessed using Spearman’s Rank correlation coefficient and significance was defined at p ≤ 0.05.Visualization of the stage analysis was performed by GraphPad Prism version 8.0.0 for Windows  quantified across 69 subjects with CKD were utilized to build the model and parameter optimization was assessed in a 3-fold cross validation setting. In brief, patients were divided into three random partitions , each time using two out of the three partitions for training, and the remaining third as a test set. Summary statistics of the classification performance for the classifier were obtained for the three folds and optimization for most suitable number of k-nearest neighbors was conducted iteratively as a function of maximizing the Area Under the Receiver Operating Characteristic curve across folds. Best fit was recorded for k = 16 neighbors and this number was further utilized to establish the final leave-one-out-crossvalidation (loocv) model. Patient specific probability scores of being labeled as deceased (scores ranging from 0.5 to 1) or censored (scores ranging from 0 to 0.5) were obtained with loocv and were converted to binary groups of “high” and “low risk”, respectively. Significant differences in survival between high and low risk groups were investigated with the Log-rank method, as described. Missing values (1.71% of the data) were replaced with the limit of quantitation for each protein (minimum concentration). Data for the knn classification were subjected to log transformation prior to training. The cox proportional hazard model was developed with the packages survival, survminer and fitting of proportionality was evaluated and examined with the functions Supplementary File.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.


Schisandra chinensis (SC) is reported to improve myocardial ischemia. Velocity vector imaging (VVI) is a noninvasive technique for evaluating myocardial function in humans, while few reported on the application in animals. In this study, we aimed to evaluate the improved effects of SC on the myocardium of Sprague Dawley rats having hyperthyroid heart disease (HHD) using VVI technique. HHD models were established by injecting daily with subcutaneous levothyroxine (0.5 mg/kg). Then, the SC group was administered the aqueous extract of SC (2 g/kg) once daily, while the HHD and control (CON) groups were administered the same amount of distilled water daily. All the rats were provided the same amount of food and water daily, and the intervention was stopped after 28 days. The efficacy of SC in HHD rats was evaluated by ultrasound VVI. The serum total triiodothyronine level, total thyroxine level, N-terminal pro-brain natriuretic peptide expression, p53 expression, and calmodulin (CaM) activity were assessed by western blotting, Hematoxylin-Eosin and Masson staining, and electron microscopy. The results indicated that SC significantly improved the systolic velocity, diastolic velocity, strain, systolic strain rate, and diastolic strain rate of the heart by significantly reducing p53 expression and CaM activity (P < 0.05), improving myocardial fibrosis in HHD rats. Also, VVI can be a valuable tool for the evaluation of myocardial function in HHD rats. Hyperthyroid heart disease (HHD) resulting from a long-term elevated level of serum thyroxine is a common complication of hyperthyroidism, causing cardiac complications such as arrhythmia, cardiac hypertrophy, or myocardial fibrosis , 2. VeloSchisandra chinensis (Turcz.) Baill (S. chinensis) is commonly known as “Beiwuweizi” in Chinese, and it has been used as a Chinese traditional herbal medicine for the treatment of many diseases. Its chemical components are mainly volatile oil, lignan, organic acids, polysaccharides, fatty oils, amino acids, pigments, and tannins. Previous studies found that Schisandra chinensis (SC) could improve myocardial ischemia and exert cardioprotective effect [e effect , 6. HereSC was purchased from the Yanji New Drug Pharmacy  and diluted in distilled water (1 : 10). After soaking for 20 min, SC water was heated, refluxed, and extracted for 45 min, twice. The extracts were freeze-dried after concentration under reduced pressure.n = 30; 5-6 weeks; 180–200 g) were provided by the Laboratory Animal Center of Yanbian University. The animal experiments were approved by the Animal Experimental Ethics Committee of Yanbian University. The rats were allowed to acclimatize for one week and, then, randomly divided into a control group , HHD group (n = 10), and SC group (n = 10). The HHD and SC groups were injected daily with subcutaneous levothyroxine (0.5 mg/kg). The CON group was injected daily with the same amount of normal saline subcutaneously. The SC group was administered the aqueous extract of SC (2 g/kg) once daily, while the HHD and CON groups were administered the same amount of distilled water daily. All the rats were provided the same amount of food and water daily, and the intervention was stopped after 28 days.Male Sprague Dawley rats  with a 10V4 probe (10 MHz). Data were saved in apical four-chamber heart sections. VVI analysis was performed offline, and the systolic velocity (Vs), diastolic velocity (Vd), strain (S), systolic strain rate (sSR), and diastolic strain rate (dSR) were obtained. VVI analysis of each rat was performed five times by a senior physician, and the mean of five experimental readings was recorded.μL serum was collected and reacted with 50 μL biotinylated antibody in the antibody-coated plates for 90 min at 37°C. Then, the microplates were washed and incubated with 100 μL streptavidin-horse radish peroxidase (HRP) for 30 min at 37°C. 3,3′,5,5′-Tetramethylbenzidine (TMB) was added and reacted for 20 min. The absorbance of the solution was measured with a microplate reader  at 450 nm.Blood samples were collected from the abdominal artery of the rats. The total triiodothyronine (TT3), total thyroxine (TT4), and NT-proBNP levels in rat serum were estimated by using ELISA kits  according to the manufacturer's instructions. In brief, 100 Proteins were extracted from frozen rat heart tissues, and the protein concentration was determined by the bicinchoninic acid (BCA) method using radioimmunoprecipitation assay lysis buffer with henylmethanesulfonyl fluoride (PMSF) . Twenty micrograms of protein were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS–PAGE) (12% acrylamide)  and transferred on to a polyvinylidene membrane. After the membrane was washed with phosphate buffered solution containing tween-20  and blocked with 5% skim milk powder solution prepared with PBST solution, it was incubated for 12 h at 4°C with the primary antibody . After the membrane was washed with tris buffered saline tween (TBST), it was incubated with the secondary antibody  at 37°C for 1 h. A chemiluminescence detection system was used to evaluate p53 expression and CaM activity. The target protein levels were normalized by GAPDH (Quantity OneVersion 4.6.2).The rats were anesthetized by injecting 20% urethane solution (0.5 mL/100 g) intraperitoneally. The hearts were excised and weighed. The left ventricular myocardial tissues were fixed in formaldehyde. The paraffin sections were stained with hematoxylin-eosin (HE) and Masson's trichrome staining kit . Four randomly selected microscopic fields from each Masson's-stained section were analyzed for collagen deposition using Image-Pro Plus 6.0 software . The results were expressed as the collagen volume fraction (CVF), which was calculated as collagen area/total area × 100%. The myocardial tissues of the left ventricular apex (1 mm³) were fixed in glutaraldehyde, processed, and observed under an electron microscope .U test (MW) (the nonparametric tests) depending on the results of the Levene test. Differences were considered statistically significant when the P value <0.05.All data are presented as mean ± standard deviation (SD) from the least three independent experiments. All data were analyzed using SPSS version 19.0 . All the data, except for the histomorphometric values, were statistically analyzed with one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The homogeneity of variance was checked using the Levene test. The histomorphometric data were statistically analyzed by either ANOVA + Tukey's post hoc test or the Kruskal–Wallis H-test + the Mann–Whitney P < 0.05). Vs and S significantly differed among the groups (P < 0.01). The Vs, Vd, S, sSR, and dSR in the SC group were higher than those in the HHD group (P < 0.05). Vs and Vd significantly differed among the groups (P < 0.01)  .P < 0.05) in serum, whereas the SC group showed decreased levels of TT3 and TT4 (P < 0.05) in serum (P < 0.05)  .P < 0.05)  .P < 0.05) compared to that of the CON group, and the CVF of the SC group decreased (P < 0.05) compared to that of the HHD group.HE and Masson's staining images of the myocardial tissues are shown in The structure of the myocardial fibers in the CON group was normal with dense mitochondria. The HHD group displayed disorders in the structure of the myocardial fibers with ruptured muscle fibers along with swollen and ruptured mitochondria. Compared to the HHD group, the SC group showed improvements in the structure of the myocardial fibers along with a reduction in the number of ruptured muscle fibers and swollen and/or ruptured mitochondria .In the present study, we investigated the feasibility of evaluating the myocardial movement in rats by using VVI. In addition, we determined the effects of SC on cardiac functions in a rat model of HHD induced by a long-term elevated serum thyroxine level. We found that the myocardial function in rats with HHD can be evaluated using VVI. Furthermore, SC was found to improve the myocardial dysfunction caused by HHD. The underlying mechanisms may be associated with the downregulation of p53 expression and CaM activity along with inhibition of myocardial cell apoptosis and myocardial fibrosis.P < 0.05). Examination of the myocardial tissues of the three groups of rats using HE and Masson's staining and electron microscopy revealed that the HHD group exhibited obvious myocardial fibrosis when compared to the CON group. This proved that the rat model of HHD was successfully established, and the findings are consistent with those of the previous studies [Persistence of elevated levels of thyroid hormones in the serum for a long time may result in cardiac overload leading to arrhythmia, cardiac hypertrophy, and myocardial fibrosis . TT3 and studies .P < 0.05). Histopathological examination revealed that the SC group showed significant improvement in myocardial fibrosis and the myocardial structure was similar to that of the CON group. Therefore, SC significantly improved the myocardial tissue structure of hyperthyroid rats.It has been demonstrated that the antioxidant, anti-inflammatory, and anti-free-radical effects of the aqueous extract of SC exert a protective effect against myocardial ischemia , 10. HowP < 0.05). Combined with the electron microscopy results, it can be suggested that the level of NT-proBNP might be used to assess the cardiac changes caused by hyperthyroidism. This result is consistent with the previous findings [P < 0.05) but showed no significant difference when compared to the HHD group. This may be attributed to the rapid accumulation of NT-proBNP in the blood, leading to its higher blood level and slower clearance.NT-proBNP is an important indicator for the diagnosis and prognosis of heart failure, left ventricular systolic dysfunction, and left ventricular hypertrophy . HHD resfindings –15. HowePrevious studies have shown that overexpression of p53, a tumor suppressor gene, induces cardiomyocyte apoptosis leading to myocardial fibrosis . In the VVI is a more objective and comprehensive technique to evaluate the myocardial function when compared to conventional ultrasonography. It reduces the dependence on angle and limitations of the section and aids in automatically tracing the trajectory of the endocardium. There are several studies using VVI for the human apical four-chamber view, although animal studies are limited to the short aorta or left ventricular long axis view –23. OwinSC fruits contain a number of compounds, such as lignans and organic acids. Schisandrin B is the most abundant lignin exerting beneficial effects on myocardial infarction following myocardial remodeling , 25. AltOur findings showed that SC significantly improved myocardial fibrosis in rats with HHD, and VVI played an important role in the evaluation of the myocardial function in these rats. SC significantly improved myocardial fibrosis in rats with HHD. VVI might be a valuable tool for the evaluation of myocardial function in these rats.


In the present study, the retinal and choroidal vascular densities (VDs) in type 2 diabetes mellitus (DM) patients were analyzed using optical coherence tomography angiography (OCTA).  The study included 282 eyes of 152 patients with type 2 DM , 48 severe NPDR, and 41 proliferative diabetic retinopathy (PDR) eyes). The superficial and deep retinal vessel, choriocapillaris, and choroidal VDs were measured using a binarization method on OCTA images. VDs were compared based on retinopathy severity. Correlations among densities were analyzed. P < 0.05). Correlation analysis showed significant positive correlations among densities of superficial and deep retinal vessels and choriocapillaris . Choroidal VD showed a negative correlation with superficial and deep retinal vessels and choriocapillaris . Retinal and choriocapillaris VDs showed a negative correlation with diabetic retinopathy (DR) grade ; however, the choroidal VD showed a weak positive correlation (P=0.030).  Retinal and choriocapillaris VDs were lower in PDR than in NPDR (all  Choroidal VD increased as retinal and choriocapillaris VDs decreased, indicating that the outer layer of the choroid is less affected by DR severity and VD of larger choroidal vessels may even be increased as a compensatory mechanism for decreased retinal and choriocapillaris VDs in type 2 DM patients. Diabetic retinopathy (DR) is a major cause of blindness in adults worldwide . AlthougVascular involvement in DR pathogenesis has previously been studied. In recent years, the introduction of optical coherence tomography angiography (OCTA) has facilitated investigations on retinal vessels as well as choriocapillaris and large choroidal vessels. An important finding in vasculature changes in DR using OCTA includes enlargement of the foveal avascular zone . In addiAlthough vascular features of the retina in diabetes mellitus (DM) patients are similar to the above-described changes, the VD change of retina and choroid and their direct correlations have not yet been clarified. In the current study, superficial and deep retinal vessel, choriocapillaris, and choroidal vessel densities in type 2 DM patients were quantitatively analyzed using OCTA and compared based on retinopathy severity. In addition, the correlation among densities was investigated.This retrospective cross-sectional case series study was performed in the Department of Ophthalmology at Bucheon St. Mary's Hospital, The Catholic University of Korea . The study was approved by the hospital's institutional review board and conducted according to the Declaration of Helsinki. Informed consent was waived due to the retrospective nature of the study.The study group consisted of consecutive type 2 DM patients who visited the ophthalmology outpatient department at Bucheon St. Mary's Hospital between December 2019 and February 2020. All patients included in the study were diagnosed with type 2 DM by doctors in the endocrinology department of the hospital. Type 2 DM was diagnosed if the patient exhibited a fasting plasma glucose level of ≥126 mg/dl or a 2 h postglucose level of ≥200 mg/dl after a 75 g oral glucose tolerance test . The sevEach patient underwent a complete ophthalmologic examination, including measurement of best-corrected visual acuity (BCVA), slit-lamp examination, and dilated fundus examination using mydriatic ultra-widefield color fundus photography  and Heidelberg HRA2 Spectralis OCTA device . DR grade was determined based on the modified Early Treatment Diabetic Retinopathy Study grade .Demographic and clinical data including age, sex, coexistence of hypertension, duration of DM, systolic and diastolic blood pressures, serum levels of random glucose, and hemoglobin A1c (HbA1c) were collected.μm or more and/or eyes with intra- or subretinal fluids) to eliminate interference of macular fluid on the vessel density analysis; (3) receiving any prior treatment for DR including anti-VEGF therapy, intraocular or periocular steroid, laser photocoagulation, or vitrectomy; and (4) presence of any significant retinal pathology other than PDR .Exclusion criteria were as follows: (1) low-quality images due to significant cataract, corneal opacities, vitreous hemorrhages, or poor cooperation and images with Q score lower than 30; (2) eyes with diabetic macular edema  of 350 En face 3 × 3 mm2 macular area OCTA images of superficial vascular complex (SVC), deep vascular complex (DVC), choriocapillaris, and choroid, centered at the fovea were obtained. Each slab was automatically obtained using predefined settings of Spectralis OCTA system. SVC consisted of nerve fiber layer vascular plexus and superficial vascular plexus, and DVC consisted of intermediate capillary plexus and deep capillary plexus. Choriocapillaris slab is generated by segmentation of 10 to 30 μm below Bruch's membrane and choroid is defined as choroidal areas below the choriocapillaris up to chorioscleral junction. Image binarization was performed using Niblack's method with a 30° radius using FIJI software . The white region was considered the vascular area in SVC, DVC, and choriocapillaris slabs and the black region was considered vascular area in choroid slab. The number of pixels was obtained and flow density was calculated by dividing the number of pixels of the vascular area by the total region of interest . For statistical analysis, the Snellen BCVA was converted to a logarithm of the minimal angle of resolution (logMAR). Values of continuous variables are presented as means ± standard deviation. One-way analysis of variance (ANOVA) was used to compare continuous variables among and between groups. Mann-Whitney and Kruskal-Wallis tests were used when a normal distribution could not be confirmed. Post hoc analysis was conducted using the Bonferroni test. Categorical variables between groups were compared using the chi-square test. Standardized adjustment was used as the post hoc test after the chi-square test. Pearson's correlation analysis was used to determine the coefficients of correlation between each VD and VDs and clinical parameters following confirmation of normal distribution. Spearman's correlation was used when normal distribution was not confirmed. Multivariate linear regression analysis was done for relevant variables. A P ≥ 0.084). DM duration was significantly shorter in patients without DR compared with other grades  and longer in patients with PDR than without DR or NPDR . Baseline demographic and clinical features of the study eyes are summarized in Of 295 consecutive eyes, we excluded 7 eyes due to poor image quality and 6 eyes due to concomitant macular diseases. In total, the present study included 282 eyes of 152 type 2 DM patients. Among the 282 eyes, 114 eyes did not show retinopathy, 79 had nonproliferative diabetic retinopathy (NPDR), 48 eyes had severe NPDR, and 41 eyes had proliferative diabetic retinopathy (PDR). All patients were Korean. The mean age was 60.12 ± 12.17 years, 57% were male, and 57% and 13% had comorbid hypertension (HTN) and chronic kidney disease (CKD), respectively. The mean duration of DM was 9.67 ± 7.91 years. The mean BCVA was 0.13 ± 0.29 logMAR. Age, sex distribution, DM duration, comorbid HTN and CKD, BCVA, random serum glucose level, and CMT did not differ significantly between DR grades . The ANOVA analysis showed that VDs of SVC, DVC, and choriocapillaris differed based on DR grade . Post hoc analysis showed that severe NPDR and PDR eyes had lower VDs of SVC and DVC compared with their previous grades .The mean VD of each layer was 0.28 ± 0.03, 0.31 ± 0.04, 0.32 ± 0.02, and 0.69 ± 0.02 for SVC, DVC, choriocapillaris, and choroidal vessels, respectively. It was 0.69 ± 0.2 for choroidal vessel measured in structural r = -0.573, −0.441, and −0.309, respectively; all P < 0.001; r = 0.129; P=0.030). Age was negatively correlated with VD of SVC . Vessel densities of SVC and DVC were negatively correlated with DM duration and comorbid HBP . The correlation between VDs and clinical parameters is summarized in B = −0.019 and −0.015; P < 0.001 and < 0.001, resp.). Age correlated with DM duration  and DM duration correlated with DR grades .Vessel densities of SVC, DVC, and choriocapillaris were negatively correlated with DR grade .Vessel densities of SVC, DVC, and choriocapillaris were strongly positively correlated  had been calculated in DR eyes and decreased CVI in PDR eyes was reported , 20. In l layers . The posl layers .The present study had several limitations. First, due to the retrospective design, factors that could affect clinical parameters, such as HbA1c and glucose levels, could not be controlled. Second, selection bias may have existed due to the exclusion of eyes with DME; however, we believe that disadvantages from excluding DME are outweighed by improved clarity of analysis. Third, the sample size of the current study was not large. Research with larger sample sizes is required to validate the results of this study. In addition, this study did not utilize an additional method for projection artifact reduction or not analyze the impact of signal strength, which can cause errors in VD analysis. Nonetheless, we minimized the error by including images with high Q score and excluding images with considerable artifacts. Also, the Spectralis OCTA system itself provides projection artifact removal. To the best of our knowledge, the association between choroidal vessel and DR grades or direct correlation among densities of retinal and choroidal vessels has not yet been reported in any study. The correlation between clinical parameters, as well as retinal and choroidal VDs, is a strength of the current study.In conclusion, choroidal VD increased as retinal and choriocapillaris VDs decreased, indicating that the choroidal large vessel layer is less affected by DR severity, and larger choroidal vessel VD may even be increased as a compensatory mechanism for decreased retinal and choriocapillaris VDs in type 2 DM patients. However, interpretation of the results should be made with caution because the correlation was not strong. We believe that the results of this study can improve the understanding of the hemodynamics associated with DR.