Background/AimsGlioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Cyclin D1 is a protein that in humans is encoded by the CCND1 gene. Cyclin D1 protein is frequently overexpressed in malignant gliomas.MethodsIt is an observational study comprising 40 biopsy-proven cases of GBM in a span of one and half years. Immunohistochemistry (IHC) was used with Cyclin D1 monoclonal antibody. Cyclin D1 on the outcome was assessed using the Kaplan-Meier survival estimate and compared by log-rank test.ResultsCyclin D1 was expressed in 60% of patients. The majority (72.5%) of patients expired during the study period, out of which 69% showed immune-expression in contrast to living subjects, out of which only 45.5% of patients exhibited expression. The maximum number of glioblastoma patients were aged between 41 and 50 years (40%), followed by those aged between 31 and 40 years (20%). The male to female ratio of study subjects was 3.44:1.ConclusionThe study concluded that there is no significant association between Cyclin D1 expression status and different demographic, clinical, and outcome variables. Glioma is a type of tumor that starts in the brain or spine. It is called a glioma because it arises from glial cells. Glioblastoma multiforme is the most common form of primary malignant tumor of the brain, and it comprises 16% of a primary brain tumor . This tuThe orderly progression of the cells through the various phases of the cell cycle is orchestrated by CDKs and their inhibitors. In humans, there are three closely related Cyclin D proteins Cyclin D1, Cyclin D2, and cyclin D3. G1/S-specific cyclin-D1 is a protein that in humans is encoded by the CCND1 . This cyIt is an observational study hospital-based conducted at the Department of Neurosurgery & Neuropathology division of the Department of Pathology, King George's Medical University, Lucknow, and Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow. The study is comprised of biopsy-proven 40 glioblastoma cases histopathologically proven cases of glioblastoma without any history of prior treatment over one and half years. The biopsies & resected specimens were collected in 10% formalin.A paraffin section was made and stained by routine hematoxylin and eosin (H&E) for histopathological examination of GBM & defined subtypes or variants of GBM. Immunohistochemistry (IHC) for cyclin D1 antigen was done using anti-cyclin D1 primary antibody and secondary antibody, and both were procured from Santa Cruz Biotechnology Inc. The primary antibody (1:100) used was cyclin D1 (DCS-6) mouse monoclonal antibody raised against the recombinant full-length human protein. The secondary antibody used was an m-IgG Fc BP-HRP  and counterstained with hematoxylin for 30 seconds.2O2 and counterstained with hematoxylin for 30 seconds. The sections were dehydrated in ascending alcohols, then dried and mounted. At every stage of the staining process, positive and negative controls were set. Immunohistochemical staining was performed using the streptavidin-biotin-HRP complex method of Immunoperoxidase Secondary Detection System (Santa Cruz Biotechnology Inc.) according to the manufacturer’s protocols.3μm-thick sections were cut from the paraffin block and placed on 3-aminopropyl triethoxysilane (APES) adhesive slides. These sections were deparaffinized at 60°C for 60 minutes, followed by incubation in xylene and hydration in a series of decreasing ethanol concentrations . The sections were placed in a pressure cooker container filled with antigen retrieval solution  and boiled for 14 minutes for antigen retrieval. The sections were washed in phosphate-buffered saline  and immersed in 3% hydrogen peroxide and 100% methanol for 10 min to remove endogenous peroxidase activity. The slides were incubated with primary antibodies at a dilution of 1:50 overnight at 4°C with cyclin D1 (clone FP12) rabbit monoclonal antibody. After washing with PBS, the sections were incubated with biotinylated goat anti-mouse immunoglobulin (Dako Ltd) for 30 min at room temperature, washed with PBS, then incubated with streptavidin-peroxidase conjugate  for 30 min. The sections were developed with diaminobenzidine tetrahydrochloride solution  and 0.1% HIHC Expression: Nuclei were scored as positive or negative in each slide. Labeling index was defined as the number of positive nuclei per total number of nuclei X 100, after counting at least 1000 cells at high power magnification. The percentage of cyclin D1 Immunopositive tumor cells was counted in 5 consecutive microscopic fields (magnification 100x) per tumor sample in the area with the highest density of these cells. One hundred tumor cell nuclei were evaluated in each field, and the mean for every five fields was calculated. A distinct granular brown nuclear stain was scored as positive. A cutoff value of <5% immunopositive cells was considered negative, and >5% immunopositive cells were considered positive. Cases categorized based on the immune expression of cyclin D1 antibody exhibited by nucleus by the tumor cell. A zero grade was given when tumor cell shows less than 5% immunoexpression of total tumor cell, low expression where immunostain shows 5 to 50% expression of tumor cells., high in more than 50% of tumor cell. Statistical tests employed are Mean, Standard Deviation, Chi-square test, Analysis of Variance (ANOVA), ROC Curve, and Level of significance.A total of 40 cases of glioblastoma were enrolled in the study. The age of cases ranged from 10 years to 69 years. The mean age of patients was 42.20±14.38 years (Median 45 years). A maximum number of patients were aged between 41-50 years , followed by those aged between 31-40 years . There were only 3 (7.5%) patients aged 21-30 years. A total of 4 (10%) patients each were aged 10-20 and 61-70 years respectively, while a total of 5 (12.5%) patients were aged between 51 to 60 years. Majority of subjects were males , there were only 9 (22.5%) females. The male to female ratio of study subjects was 3.44:1. A total of 37 (92.5%) patients were GBM alone. There were three patients (7.5%), each having GBM with small cell, Giant cell GBM and Gliosarcoma respectively. The temporal site was the most involved, followed by parietal (25%), frontal (20%), and occipital (20%) parts, respectively. Cyclin D1 was expressed in 24 (60%) patients. The majority of subjects (23) had expression in the range of 5-50%. Sixteen (40%) subjects had no expression, while 1 (2.5%) patient had high expression.  . One value (66%) has been depicted as an outlier on the plot and excluding this value, the range of expression becomes from 0% to 38% , and the median was 10%. The 5th to 95th percentile range of expression was between 0 to 37.70%. Survival analysis for subjects with different Cyclin D1 expression strata revealed that though the median survival time was higher among those with no Cyclin D1 expression (259.00±102.21 days) as compared to those with Cyclin D1 expression (217.00±6.79 days) yet the difference between the two strata was not significant statistically (p=0.120 - Log Rank - Mantel-Cox test) . However, as compared to those who expired the mean expression percentage was significantly higher among those who survived p=0.035). As a significant association between survival and mean expression percentage was observed, a receiver-operator curve (ROC) analysis was done to find a suitable cutoff to predict the survival outcome based on the expression percentage of Cyclin D1. The area under the curve was observed to be 0.715, which indicated a moderate efficacy of the percentage of Cyclin D1 expression to predict the outcome expiry. A cutoff value >10.50 was observed to be 55.2% sensitive and 81.8% specific , followed by the parietal region (25%). Twenty percent 20%) of the tumors involved the frontal and the occipital regions. This was in close association with the study by Lee TT et al., 1997 at University Hospital Zurich in which the temporal lobe was involved in 31% of patients, the parietal lobe in 24%, frontal in 23%, and occipital lobe in 16% of the patients [% of the In the present study, most subjects had expressions in the range of >5-50% were considered the low expression, only 2.5% had >50% expression was considered the high expression, and the remaining 40% subject had no expression. Cyclin D1 is a protein encoded by the CCND1 gene and is a cell cycle regulator at the G1-S checkpoint. In our study, cyclin D1 was expressed in 62.5% of patients with GBM. Its mean value among 40 patients was 11.85±13.944, and the median value 10.00. As per the study by Zhang X et al., 2005 cyclin D1 was expressed in 55.8% (29 of 52) gliomas . BiernatBiernat W et al. and Mahzouni P et al. showed that Cyclin D1 is a wrong prognostic marker of GBM. Slight staining on IHC for cyclin D1 is found in normal brain and tumors of WHO Grade-1 ,23. WithOur findings are similar to those of Ahmad F et al. (2009), who observed staining of that cyclin D1 was highly expressed in benign and low grades gliomas of brain tumors nevertheless decreased in higher grades of tumors . KrtolicSimilarly, Chakrabarty A et al. (1996) found a strong positive correlation between labeling indices for cyclinD1and MIB1 proliferation marker in diffuse astrocytoma . CorrelaMahzouni P et al. observed cyclin D1expression only in normal brain tissue but marked overexpression of cyclin in glioma . In our Similar to the study done by Tan PG et al. (2004), the cyclin D1 positive ratios were 66.67% 14/21) and 32.00% (8/25) in the dead group and survival group, respectively [/21 and 3Most patients expired during the study period, within one year of diagnosis. No significant association between Cyclin D1 expression status and different demographic, clinical, and outcome variables could be observed. Survival analysis for subjects with different Cyclin D1 expression strata revealed that the difference between the two strata was not significant. There was no association between age, gender, and diagnostic entities with cyclin D1 expression. The mean immuno-expression of Cyclin D1 was significantly higher among those who expired than those who survived. ROC curve indicated a moderate efficacy of the immuno-expression of Cyclin D1 to predict the outcome expiry. A maximum number of glioblastoma patients were aged between the fourth and fifth decades. There is male predilection. Most patients were GBM alone, and the temporal site was the most involved, followed by the parietal.


In situ hybridization (ISH) is a complementary technique to sequencing and culturing to visualize the presence of individual bacterial operational taxonomic unit (OTUs) in context of the colonized organ. We recently applied highly sensitive in situ RNA hybridization to detection of commensal bacteria in low abundance respiratory tract samples of mice housed under specific pathogen free conditions. This technique allows species-specific detection of living bacteria using RNAScopeTM technology, while preserving the natural environment of the organ. We here provide a detailed step-by-step protocol describing the detection of commensal lung bacteria in respiratory tissue.Commensal microbes are an integral component of mammalian physiology. 16S rRNA gene-specific next generation sequencing from DNA of total organs, swabs or lavages has revolutionized the characterization of bacterial communities in virtually every ecological niche of the body. Culturomics, next allowed the isolation and characterization of commensal bacteria in the lab and the establishment of artificial communities of bacteria, which were eventually reintroduced in model organisms. Spatial organization of microbiota within a given host environment is critical to the physiological or pathological phenotypes provoked by commensal microbiota.  Previous studies have used fluorescent based approaches to determine spatial organization of commensal microbiota in the intestine and lung , micro-dissecting forceps (F.S.I.) Falcon 15-mL conical centrifuge polypropylene tubes (Thermo Fisher Scientific), Paraformaldehyde 4% (Merck), Ethanol 70%.Cooling block grid (Leica), histology cassettes (Leica) microscope slides (Thermo Fisher Scientific), microtome (Leica RM2235), Paraplast (Leica), Ethanol with 2% methyl ethyl ketone (MEK) (Biosystems), Histo-Sav .Histological grade xylene, ethanol 99.9 grade, Fume-hood, x8 50-mL beakers, dry oven.RNA Scope kit (ADC) containing: RNAScope Hydrogen Peroxide, RNAScope 10X Target Retrieval, RNAScope 50X Wash Buffer, RNAScope Protease Plus, RNAScope Hydrophobic pen, RNAScope probes (#475131 or #451961), RNAScope AMP1-6 Reagents, RNAScope Fast Red-A, RNAScope Fast Red-B, RNAScope EcoMount. Lysozyme (40 μg/mL), 5X SSC Buffer , Meyer’s Solutions (Sigma), Ammonia, Histological grade xylene, Steam Cooker , Coverslips, Dry Oven, HybEZ Oven, Slide’s Rack for washing.Brightfield Olympus BX61VS120 with a motorized stage (Olympus LifeSciences) equipped with Plan Apo N 2×/0.08 and U Plan S Apo 100×/1.4 Oil objectives and a Pike F505C VC50 detector (Allied Vision Technology).This methodology was adapted from the protocol guidelines provided by the ACD RNAScope platform . Furtherad libitum. Seven to eight weeks colonized C57BL/6J mice were housed in a specific pathogen-free and climate-controlled animal facility at the University of Geneva. Mice were provided with water and pelleted diet ad libitum. All cages were provided with environmental enrichment in the form of nesting material and mouse houses. Procedures and experimentation were carried out in accordance with federal regulations of the Bundesamt für Lebensmittelsicherheit und Veterenärwesen (BLV) Switzerland (Tierschutzgesetz) and approved by an institutional review board and the cantonal authorities. Animal license GE/159/17.Germ free animals with C57BL/6J background were generated and maintained by the Clean Mouse Facility of Department of Biomedical Research of the University of Bern kindly provided by Dr. Mercedes Gomez and Prof Sigfried Hapfelmeier. They were born and raised in flexible film isolators in at the University of Bern, transferred aseptically into sterile IVC cages provided with sterile food and water 1.12 exposure). Confirm death by controlling from absence of reflex after pinching the footpad of the hind limbs.Euthanize animals  and stored at room temperature for 24 h. Make sure that the organ is fully immersed in the fixative. This can be achieved e.g., by adding the lid of a 15 ml tube upside down into the 50 ml tube, to hold the lung below the air-liquid interface. Of note, inflation of the lung with fixative is not recommended, since it could flush commensal bacteria out of their natural niche and might lead to artificial location in the organ.2.1After fixation place lungs in cassettes, orient them properly according to the envisioned cutting program and dehydrate as follows.2.2Immerse lungs fully in 70% ethanol for 2 h at RT.2.3After 2 h Immerse Lungs fully a second time in 70% ethanol for 2 h at RT.2.4Immerse lungs fully in 90% ethanol for 1 h at RT.2.5Immerse lungs fully in 95% ethanol for 1 h at RT.2.6Immerse lungs fully in 100% ethanol for three consecutive steps of 30 min.2.7Transfer lungs into Histo-SAV I  for 30 min.2.8Immerse lungs fully in Histo-SAV II  for 30 min.2.9Immerse lungs fully in Histo-SAV III  for 30 min.2.10Transfer lungs into paraffin I for 2 h.2.11Transfer lungs into paraffin II for 4 h.2.12After hardening, cut paraffin blocks into 1 μM frontal sections using a microtome (Leica RM2235 Rotary Microtome).2.13Perform five consecutive transversal cuts at four different depths . Cutting could be adapted according to experimental goals.2.14Transfer two consecutive slices of each depth onto one microscope slide and leave them to dry overnight at RT. Slides can be left either unstained or stained e.g., with Giemsa.2.15Submerge the slides in 20% Giemsa dye  diluted in distilled water.2.16Quickly dip the slides in 0.2% acetic acid (Sigma Aldrich) diluted in distilled water (organ samples should turn pink).2.17Quickly dip the slides in ethanol 90% (organ samples should turn blue).2.18Submerge the slides in isopropanol (Fluka) for 2 min.2.19Quickly dip the slides in ethanol 100%.2.20Quickly dip the slides in Histo-Sav .2.21Mount the slides following the protocol provided by UltraKit .2.22Place the slides containing the organ cuts in a dry oven for 1 h at 60°C to dry.2.23Place eight 50-mL beakers under a fume-hood and filled with either histological grade xylene or ethanol 99.9 grade (four beakers for each solution).x2.24Dip the dried microscope slides first into each xylene-containing beaker for 5 min each.2.25Continue with the ethanol-containing beakers for 1 min each and leave to air dry. Deparaffinized slides were used for the RNAScope procedure described in the next section see also .3.1Target Retrieval3.1.1Lay the slides in the HybEZ tray and add enough drops of RNAscope Hydrogen Peroxide to cover the samples on each slide.3.1.2Incubate at room temperature for 10 min.3.1.3Tap the slides on top of absorbent paper to remove excess liquid and immediately submerge the slides in a reservoir containing distilled water.3.1.4Lift the slides up and down for a proper wash.3.1.5Repeat the washing step.3.1.6Place two reservoirs containing RNAscope 1X Target Retrieval Reagent and Distilled water in a steam cooker  machine set at 99°C.3.1.7Add slides to the distilled water container for 10 seconds and move them into the container with RNAscope 1X Target Retrieval Reagent.3.1.8Monitor the temperature with a thermometer.3.1.9This incubation time is susceptible to changes according to the samples.Cover the steam cooker and incubate for 45 min refer to . This in3.1.10Meanwhile, pre-warm HybEZ Oven at 40°C for 10 min.3.1.11Remove slides from steam cooker and wash with distilled water.3.1.12Move the slides to a container with Ethanol 99.9 grade for 3 min.3.1.13Dry the slides in a dry oven at 60°C.3.1.14Using the hydrophobic pen, draw a circular barrier around the samples on each slide and let it dry completely at room temperature for 5 min refer to .3.1.15Lay the slides in the HybEZ tray and add enough drops of RNAscope Protease Plus to cover the samples on each slide.3.1.16This incubation time is susceptible to changes according to the samples.Incubate at 40°C inside the tray of the HybEZ Oven for 30 min. 3.1.17Tap the slides on top of absorbent paper to remove excess liquid and immediately submerge the slides in a reservoir containing distilled water refer to .3.1.18Lift the slides up and down for a proper wash.3.1.19Lay the slides in the HybEZ tray and add enough drops of Lysozyme (40 μg/mL) to cover the samples on each slide .3.1.20Incubate at 40°C inside the tray of the HybEZ Oven for 90 min .3.1.21Tap the slides on top of absorbent paper to remove excess liquid and immediately submerge the slides in a reservoir containing distilled water.3.1.22Lift the slides up and down for a proper wash.3.2.1Pre-warm the HybEZ Oven at 40°C and the RNAScope probes at 37°C for 10 min.3.2.2Tap the slides on top of absorbent paper to remove excess liquid and lay them in the HybEZ tray.3.2.3Add enough drops of the appropriate probe to cover the samples on each slide refer to .3.2.4Incubate at 40°C inside the tray of the HybEZ Oven for 2 h refer to .3.2.5One at a time, tap the slides on top of absorbent paper to remove excess liquid and place it back on the tray.3.2.6Submerge the slides in a container filled with 1X Wash Buffer and lift them up and down for a proper wash.3.2.7Repeat the washing step.3.2.8Optional Step: Place the slides in a container with 5X SSC Buffer and leave them overnight.3.2.9Remove excess liquid by tapping the slide on top of absorbent paper and place them back on the tray.The next steps consist on the amplification of the probe signal by adding AMP1-4 reagents and incubating them at 40°C inside the tray of HybEZ Oven at 40°C.Note: Always add enough drops of each reagent to cover the samples on the slides and remove excess liquid after incubation.3.3.1Add AMP 1 Reagent and incubate for 30 min refer to .3.3.2Wash the slides twice with 1X Wash buffer.3.3.3Add AMP 2 Reagent and incubate for 15 min.3.3.4Wash the slides twice with 1X Wash buffer.3.3.5Add AMP 3 Reagent and incubate for 30 min.3.3.6Wash the slides twice with 1X Wash buffer.3.3.7Add AMP 4 Reagent and incubate for 15 min.3.3.8Wash the slides twice with 1X Wash buffer.The next steps consist on the final amplification of the probe signal by adding AMP5-6 reagents and incubating them at room temperature.3.3.9This incubation time can be modulated to adjust probe signal intensity.Add AMP 5 Reagent and incubate for 30 min. 3.3.10Wash the slides twice with 1X Wash buffer.3.3.11Add AMP 6 Reagent and incubate for 15 min.3.3.12Wash the slides twice with 1X Wash buffer.3.3.13Meanwhile, prepare a solution of Fast RED (A + B) by adding: 1 volume of Fast RED-B and 60 volumes of Fast RED-A.3.3.14Pipette enough volume of Fast RED mix to cover the samples in each slide (∼300 μL per slide) and incubate for 10 min at room temperature.3.3.15Tap the slides on top of absorbent paper to remove excess liquid and lay them in the HybEZ tray.3.3.16Rinse the slides with distilled water.4.1This dilution can be modulated to adjust staining intensity.Submerge the slides in a container filled with Meyer’s solution diluted 1:2 for 2 min at room temperature refer to  This dil4.2Wash the slides with distilled water until the water is clear and has no traces of Meyer’s solution.4.3Submerge the slides in 0.02% ammonia diluted in distilled water and move the slides up and down a few times until the sample turns blue.4.4Wash the slides with distilled water.4.5Dry the slides at 60°C for at least 15 min or air dry until all visible liquid is evaporated.4.6Briefly dip the slides into a 50-mL beaker containing fresh histological grade xylene and add 1–2 drops of EcoMount (Vectamount) while the slides are still wet.4.7Cover the sample with a coverslip carefully to prevent formation of air bubbles and let it air-dry.A large variety of imaging systems can be used for visualization. The following steps are thus solely to indicate a technical guidance and should be adapted to the existing infrastructure in each laboratory.5.1Visualize slides using a Olympus VS120 brightfield microscope (Olympus) and QuPath-0.2.1 Software.5.2Screen slides first with a 2× magnification for a quick visual scan of the overall distribution of positive signal (red-pink staining).5.3Choose a one random field from each slice and scanned again with the 100×/1.4 Oil objective using a multifocal approach. The VS-ASW creates a focus map with multiple coordinates defined by the user. This allows the optimal Z-position to be determined and saved on various parts of the sample, allowing a height profile of the sample to be compiled before detailed scan acquisition.5.4This value can be adapted to the experimental requirements of each user.Process and analyze an average of 5–8 fields per sample at higher resolution. 5.5Determine positive staining of red-pink punctate dots around epithelial cells of airway ducts.In order to get an impression of the quality of tissue slices we performed Giemsa staining .Technically this should allow the detection of bacteria in tissue , howeverGenerally, the consultation of experienced histo-pathologists is advised in order to evaluate the quality of tissue slices.L. murinus used here will provide a bright red-pink coloration of bacterial clusters (black arrows) attached to the medium or large airways  contains about 10bia agar , most of•It is important to always use freshly prepared histological grade xylene, ethanol 99.9 grade and Meyer’s solution.•The efficiency of the staining process primarily depends on the target retrieval step, which allows the permeabilization of the tissue granting the probe access to the target.•The temperature of the steam cooker is crucial to be maintained constant (between 96 and 99°C) throughout the target retrieval incubation. The use of a thermometer on the steam cooker helps monitoring the temperature with the specified machine.•L. murinus are known to have a thicker cell wall. For that we introduced a lysozyme incubation step for 90 min at 40°C. However, this additional exposure did not improve staining results (data not shown).We tried an additional stage of permeabilization since gram-positive bacteria such as •Another important point during the staining process is the adjustment of the time of incubation during the signal amplification step. Amp5 incubation can be left for longer or shorter times for intensity regulation of the probe signal. On the same note, Meyer’s solution dilution can also be adjusted for a stronger counterstaining of the tissue slice. The fine-tuning of both these steps is helpful if the probe intensity is faint and contributes to a better contrast between counterstaining and target signal.In this article we presented a general pipeline for ISH allowing visualization of commensal bacteria in an environment of low-colonization density. Combined with specific probes, detection of a given bacterial species in context of its natural organ context is possible. The technique provides a high signal to noise ratio as shown by the lack of background staining in axenic animals. In contrast to hybridization approaches targeting bacterial DNA , it furtWe chose a colorimetric approach in order to better visualize the proximity to the host tissue , but in L. murinus in the lung of a SPF housed mouse as 107, based on specific qPCR data (In a recently published study we estimated the total genome copy number of PCR data . NGS datAcidovorax as a potential biomarker for lung cancer (An interesting future expansion of this technique would be the combined detection of bacterial RNA and host RNA, to visualize, e.g., host responses of cells in the direct environment of commensal or pathogenic bacteria. Using distinct fluorescently labeled probes, such a staining is perfectly in accordance with the here proposed techniques and would require only little adaptation. In context of cancer diagnostics, a similar approach was used to confirm the presence of the bacteria strain g cancer . This apg cancer .A combination of several probes for the detection of multiple bacterial species in more complexly colonized animal would equally be of interest. For the intestinal tract FISH was already used to establish a biogeographical map indicating microbiota distribution on phylum level . In miceIn combination with 16S specific rRNA gene NGS and metagenomic shot gun approaches we propose the use of complementing FISH or ISH approaches to localize commensal bacteria of interest in organ context.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Direction de l’expérimentation animale, University of Geneva and SCAV Canton Geneva.JPPB performed the ISH experiments and optimized the technique for detection of bacteria in lung tissue. Both authors wrote the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human bocavirus (HBoV) is globally distributed and associated with respiratory and enteric infections. Limited data are available about the incidence of HBoV in Egyptian children. We aimed to investigate the association of HBoV genotypes in children with diarrheal disease and also to determine the possibility of HBoV co-infections with other human enteric pathogens.A total of 102 stool samples were collected from children under five years old with diarrhea. Samples were analyzed for the presence of HBoV by real-time PCR. HBoV positive samples were tested for adenovirus (AdV), rotavirus (RoV), parasitic helminths, and enteric protozoa.4 GC/g in males and 1.03 × 105 GC/g in females. Among the HBoV-positive samples, the single infection of HBoV was 52.5% (31/59), while the co-infections with multiple viruses were found in 1.7% (1/59) for HBoV and AdV, 33.9% (20/59) for HBoV and RoV, and 11.9% (7/59) for HBoV, and RoV and AdV. No co-infection with parasitic helminths or enteric protozoa was found.HBoV was detected in 58% of examined cases. HBoV-3 was the most prevalent genotype observed , followed by HBoV-2/4  and HBoV-1 . Although the incidence of HBoV was higher in males  than females , the analysis showed no significant difference for HBoV between genders. The average HBoV concentrations were 5.3 × 10The single infection of HBoV in some children suffering from acute gastroenteritis indicated that HBoV could be the main etiologic agent of the disease. The study highlights the high incidence of HBoVs genotypes with remarkable multiple co-infections in the pre-school children in Egypt. Diarrheal disease is the second most common cause of mortality worldwide in children less than 5 years and it is estimated that 600,000–700,000 infants and young children die from diarrhea each year. Most of the deaths occur in Sub-Saharan Africa and South Asia and mortality is high in children less than 5 years . MortaliHuman bocavirus (HBoV) was discovered in 2005 . HBoV isA plethora of studies detected HBoVs in Europe , North AHBoVs cause a variety of clinical manifestation and could be isolated from several types of clinical samples including nasopharyngeal aspirates , 16 and The association of HBoV with respiratory disease and acute gastroenteritis remains unclear due to the high rate of co-infection with other pathogenic viruses in symptomatic patients, as well as its frequent detection in asymptomatic individuals , 21. HowA total of 102 stool samples were collected anonymously from preschool children  from private clinics in Giza, Egypt. The samples were collected from children under 5 years of age suffering from acute watery diarrhea. Stool samples were examined immediately at the same day for parasites identification. In order to avoid repeated freezing and thawing, stool samples were aliquoted, stored at − 20 °C, and tested within a month from collection for HBoV.About 100 mg of fecal diarrhea samples were weighed and diluted in phosphate buffer saline (1:10). The samples were vortexed for 30 s followed by centrifugation at 5000 rpm for 10 min at room temperature. The supernatants were kept at − 80 °C until further use.Viral nucleic acids were extracted from 200 μl of the concentrated sample using GeneJET Viral DNA and RNA Purification kit (Thermo Scientific-USA) according to the manufacturer’s instructions. The obtained nucleic acid was dissolved in 60 μl of eluent and kept at – 80 °C until use.NanoDrop Fluorospectrometer . The DNA concentration was converted to genomic copies using the following formula: number of DNA copies = (DNA amount (ng) × 6.022 × 1023)/(length (bp) × 1 × 109 × 650).All primers used in the current study were listed in Table 1 to 5 × 107 copies/reaction. For HBoV-1 and HBoV-3, the slop was − 3.69; the coefficient of determination (R2) was 0.99. In the case of HBoV-2/4, the mean value of the slope was − 3.45; the mean of R2 was 0.99. Virus concentration per gram (g) GC/g was calculated according to the following equation: GC/g = GCxDF× 10, where GC is genome copy number per reaction, DF is the dilution factor for the volume reductions that occur during the concentration, DNA extraction, and qPCR steps, and the obtained GC was expressed per gram of stool sample.The standard curve of each bocavirus was separately prepared by tenfold serial dilution of the nucleic acid standard ranging from 5 × 10The positive samples for HBoV were tested for RoV and AdV to determine the pathogen co-infections. RoV was detected by using nested RT-PCR for the detection of the VP6 segment . AdV wasThe positive samples for HBoV were tested for enteric protozoa and helminth parasites by microscopic examination (direct wet-mount preparation)  to diffeP value for the test was set at < 0.05. One-way ANOVA shows the significant difference between the relative distribution of different bocavirus genotypes in total, male and female stool samples. The unpaired t test was used to compare each HBoV genotype in male versus female stool samples.Statistical analyses were performed using GraphPad Prism version 8.3.0 software (USA). The critical P < 0.05) of the children stool samples using qPCR, which targets NP-1 and NS-1 genes. Statistically, the viral type showed a significant effect on the prevalence of HBoV in males (P = < 0.0001), although no significant influence on the prevalence of the virus in females (P = 0.27). The detection rates of different HBoV genotypes are presented in Table In the present study, human bocavirus genotypes were detected in 58% . In other words, gender had no significant effect on the prevalence of HBoV-1. The average viral concentration was 1.0 × 104 GC/g in males and 1.3 × 104 GC/g in females . The average viral concentrations were 1.3 × 105 GC/g and 2.6 × 105 GC/g in males and females, respectively  than females (27.5%)  in total samples compared to other HBoV types. The prevalence of HBoV-3 was higher in males (49 %) than females 39.2%)  of HBoV positive cases and two or more genotypes detected in 66% (39/59) of HBoV positive cases. The most common mixed genotype cases were HBoV-1 and HBoV-3 (20.3%), followed by HBoV-2/4 and HBoV-3 (18.6%) and HBoV-1 andHBoV-2/4 (6.7%) (Table 0/59 of HThe positive samples for HBoV (n = 59) were screened for other viruses , protozoa, and parasitic helminths to explore whether AGE originated from bocavirus or other causative agents. The single infections of HBoV in males and females were 61.8% 21/34) and 40% (10/25), respectively, while the co-infections ratios of HBoV/AdV, HBoV/RoV, and HBoV/RoV/AdV in males were 2.9 % (1/34), 26.5% (9/34), and 8.8% (3/34), respectively. In females, multiple infections of HBoV/RoV and HBoV/RoV/AdV account for 44% (11/25) and 16% (4/25), respectively  was higher than co-infection with AdV and RoV, indicating the possible contribution of the virus in the pathogenicity. In contrast, infection with HBoV in Pakistani children was not significantly associated with gastroenteritis alone where 98% of HBoV reported cases had co-infection with RoV . The co-In the present study, the prevalence of HBoV in children suffering from AGE was 58% which is higher than the previous reports of AGE associated with HBoV in Egypt (2%) , Brazil p > 0.05). The current results agree with the study of Nawaz et al. [In the present study, the viral incidence was higher in males 66.6% than females 49% and the difference was statistically non-significant .The presence of HBoV in some children suffering from AGE without the association of any other etiological agents  indicates the ability of the virus to cause the disease. HBoV was abundant in stool samples from children with gastrointestinal disease in Egypt. Higher infection rates were detected in males rather than females. However, the viral loads were higher in females than in males. HBoV-3 was the most abundant among HBoV genotypes. Higher proportions of multiple co-infection of HBoV genotypes were recorded compared to single infections. The viral type had a strong significant effect on the prevalence of HBoV rather than human gender. Moreover, the concentration of HBoV-2/4 was higher than HBoV-1 as well as HBoV-3. Taken together, the data obtained in our study raise a concern on the role of HBoV in gastrointestinal illness.


RIPC was a significant independent variable for lower overall postoperative morbidity survey (POMS) score, OR 0.79 (95% CI 0.63 to 0.99) and fewer complications at 6 months including pulmonary OR 0.2 (95% CI 0.03 to 0.92), surgical OR 0.12 (95% CI 0.007 to 0.89) and overall complications, OR 0.18 (95% CI 0.03 to 0.74). There was no difference in perioperative troponin change or TIMP2*IGFBP-7. Our pilot study suggests that RIPC may improve outcomes following intra-abdominal cancer surgery and that a larger trial would be feasible.There is limited evidence on the effect of remote ischaemic preconditioning (RIPC) following non-cardiac surgery. The aim of this study was to investigate the effect of RIPC on morbidity following intra-abdominal cancer surgery. We conducted a double blinded pilot randomised controlled trial that included 47 patients undergoing surgery for gynaecological, pancreatic and colorectal malignancies. The patients were randomized into an intervention (RIPC) or control group. RIPC was provided by intermittent inflations of an upper limb tourniquet. The primary outcome was feasibility of the study, and the main secondary outcome was postoperative morbidity including perioperative troponin change and the urinary biomarkers tissue inhibitor of metalloproteinases-2 and insulin-like growth factor-binding protein 7 (TIMP-2*IGFBP-7). The recruitment target was reached, and the protocol procedures were followed. The intervention group developed fewer surgical complications at 30 days (4.5% vs. 33%), 90 days (9.5% vs. 35%) and 6 months (11% vs. 41%) (adjusted  Morbidity following cancer surgery may affect both short- and long-term outcomes resulting not only in prolonged hospital stay and delayed adjunct treatment but also in decreased survival and disease recurrence ,2. Body This was a single-centre double-blinded interventional pilot study with randomization 1:1 into intervention (RIPC applied to an upper limb) or control (tourniquet applied but not inflated). The study was approved by the London-Fulham NRES Committee  on 26 October 2018. The protocol was registered at the CPMS (ID 38780) and ISRCTN registries (ISRCTN 11439947). A sample size of 50 participants was targeted as big enough to allow assessment of the study procedures and power analysis for a substantive trial. The study was conducted at Royal Surrey County hospital from May 2019 to April 2020. Written informed consent was obtained. A web-based randomisation system was used with block sizes of 4 and 6 and stratified per surgical specialty. The randomisation outcomes were kept in sequentially numbered opaque sealed envelopes that were opened after induction of anaesthesia. The participants, the clinical teams looking after them and the data collectors were blinded as to the whether RIPC was administered or not. The anaesthetists responsible for the intraoperative care were not blinded, as this was not practically possible.Patients eligible to take part in the study were adults undergoing elective surgery for colorectal, pancreatic or gynaecological cancer under general anaesthesia and with an estimated morbidity risk of at least 10%, as predicted by the American College of Surgeons National Surgical Quality Improvement Project (ACS NSQIP) tool . The excParticipants allocated to the intervention group received RIPC by a member of the research team after the induction of general anaesthesia and prior to skin incision. RIPC was provided by means of inflation of an appropriately sized cuff of an automated tourniquet machine placed on an upper limb and inflated at a pressure of 200 mmHg for 5 min at a time for a total of three times with 5-minute intervals of no inflation. Participants randomized to the control group had the tourniquet placed at an upper limb but not inflated. ®), hospital length of stay and quality of life. TIMP-2 and IGFBP-7 are inducers of G1 cell cycle arrest *[IGFBP-7])/1000 (ng·mL−1)2. A single cut-off between 0.3 and 2 (ng·mL)2/1000 has been shown to be associated with at least 4 times increased risk of developing moderate-severe AKI (KDIGO stage 2–3) in the following 12 h *[IGFBP-7] biomarkers was collected 4 h following the end of the operation. The samples were centrifuged, and aliquots were stored at −70 °C with the urine samples after freezing on dry ice. Following participant recruitment, the serum samples were thawed and analysed using electrochemiluminescence immunoassay (Siemens ADVIA CentaurPostoperative morbidity was assessed using the postoperative morbidity survey (POMS) on days 2, 3 and 5  providedThe feasibility of the study included the assessment of the proportion of potentially eligible patients that were recruited to the study and followed up at the specified timepoints and the proportion of blood and urine samples collected and analysed.t-test or Wilcoxon’s signed rank test as appropriate, following the Shapiro–Wilk test for normality. Categorical variables were analysed using the χ2 or Fisher’s exact test. Where categorical variables were assessed at three time points, the Cochran–Mantel–Haenszel χ2 test was used followed by groupwise analysis with time as the grouping variable and false discovery rate (fdr) adjusted p values. Linear and binomial regression analysis were used to assess the potential association of any independent factors with continuous and categorical outcomes, respectively, at the studied time points. The independent variables were chosen based on clinical judgement and the previous literature. The independent variables used in the binomial regression for POMS morbidities were age, American Society of Anaesthesiologists (ASA) grade, risk of overall postoperative morbidity as predicted by ACS NSQIP, treatment group, surgery and perioperative troponin change. In addition to the above, preoperative cardiovascular (CVS) disease and estimated blood loss (EBL) were included in the independent variables of the regression analysis for CVS morbidity, preoperative pulmonary disease for pulmonary morbidity and chronic kidney disease and urinary biomarkers for postoperative AKI. The independent variables used in the binomial regression for ACS NSQIP-defined complications were age, ASA grade, ACS NSQIP-predicted risk, treatment group, surgery and perioperative troponin change. Additionally, preoperative CVS disease and POMS CVS morbidity were included in the independent variables for postoperative ACS-defined CVS complications, preoperative pulmonary disease and POMS pulmonary morbidity for ACS pulmonary complications, POMS gastrointestinal morbidity for ACS gastrointestinal complications, and chronic kidney disease and Nephrocheck result for ACS urinary complications. Binomial regression for each type of POMS morbidity and ACS complication was performed with Firth’s correction. Backward elimination from the initial model was used in order to establish the best fit model. Results were considered significant if the p value was less than 0.05. Statistical analysis was conducted using R  and figures were produced using the package ggplot2  4 ng·L−1  and postoperative hs-TropI was 6 ng·L−1  (p = 0.186). Only one patient had postoperative hs-TropI above the upper reference limit; however, the patient’s preoperative hs-TropI was also above the upper reference limit. There was no difference in the perioperative troponin change between the intervention and control groups, median [IQR] 1  vs. 1 , respectively (p = 0.617). Linear regression analysis of perioperative troponin change with independent variables age, preoperative CVS disease, ASA grade, ACS NSQIP predicted morbidity, EBL and surgery showed that age and surgery were the significant variables, with gynaecological and pancreatic surgery and increasing age being associated with greater troponin change, with odds ratio (OR) of 2.29 (95% CI 1.40 to 3.75), 2.73 (95% CI 1.58 to 4.72) and 1.04 (95% CI 1.01 to 1.06), respectively.Overall CVS POMS morbidity was 13/24 (54%) vs. 17/22 (77%) in the intervention vs. control groups (p = 0.006). However, further analysis of the type of morbidity showed that the difference was in the presence of urinary catheter rather than the development of AKI on day 5. The overall incidence of AKI was 7/47 (15%) in hospital and 9/47 (19%) within 6 weeks postoperatively. Four of these patients underwent colorectal surgery, one gynaecological and four pancreatic. Four of the 24 patients (16.67%) in the intervention group and five of the 23 in the control group (21.7%) developed AKI (p = 0.724). Of the seven in-hospital cases of AKI, five developed in the first 24 h, one at 24–48 h and one on day 7. Logistic regression of in-hospital AKI showed that troponin change was significant, OR = 12.29 (95% CI 1.82 to 219.70). TIMP-2*IGFBP-7 at 4 h postoperatively was 0.56  vs. 0.21  in patients with and without AKI in the following 12–24 h (p = 0.213). However, four of the five (80%) cases of AKI in the first 24 h were stage 1 and only one (20%) was stage 2. There was no difference in TIMP-2*IGFBP-7 values between the treatment groups, 0.19  vs. 0.34 , intervention vs. control, (p = 0.1016). TIMP-2*IGFBP-7 was ≥0.3 in 5/20 (25%) vs. 12/20 (60%) of patients in the intervention and control groups, respectively (p = 0.054). There was significantly less renal morbidity in the intervention group on day 5 (adjusted p = 0.232). Logistic regression of any POMS pulmonary morbidity showed that only surgery was significant with an OR of 25.62 (95% CI 3.41 to 366.87) for pancreatic surgery and 7.1 (95% CI 1.46 to 48.04) for gynaecological surgery.The overall incidence of pulmonary POMS morbidity was 10/24 (42%) vs. 14/22 (64%) in the intervention vs. control groups (p = 0.346). Logistic regression for any gastrointestinal morbidity showed that only surgery was significant with an OR of 10.03 (95% CI 1.68 to 110.71) for pancreatic and 3.69 (95% CI 0.93 to 16.11) for gynaecological surgery.The overall incidence of POMS gastrointestinal morbidity was 15/24 (63%) vs. 17/22 (77%) in the intervention vs. control group (p = 0.217). The treatment group was not an independent predictor of pain morbidity in univariate analysis. The incidence of other types of POMS morbidity was overall small and is described in Overall pain morbidity was 14/24 (58%) vs. 17/22 (77%) in the intervention vs. control group .Length of stay was 5 [3.5 to 8.5] vs. 7 [4.25 to 8] in the intervention vs. control group, The incidence of any ACS complication is shown in p of 0.165, 0.062 and 0.062 at 30 days, 90 days and 6 months, respectively). Logistic regression of any ACS postoperative complication showed that the treatment group was significant at day 90 and 6 months with an OR of 0.28 (95% CI 0.07 to 0.99) and 0.18 (95% CI 0.03 to 0.74), respectively.Groupwise analysis did not show a difference between the two groups (adjusted p = 0.646). Logistic regression for day 30 CVS complications showed that the presence of in-hospital POMS CVS morbidity was significant, OR 11.63 (1.54 to 221). For day 90 and 180, preoperative cardiovascular morbidity was significant, OR 5.13 (95% CI 1.18 to 26.15) and OR 6.4 (95% CI 1.3 to 40.78), respectively.There was no difference in the incidence of cardiovascular complications between the two groups (p = 0.947). Within 6 months in the intervention vs. control group, 3/18 (16.67%) vs. 2/14 (14.29%) patients were treated for a urinary tract infection, 4/24 (16.67%) vs. 5/23 (21.74%) had AKI and 0 vs. 1/15 needed renal replacement therapy. Logistic regression of urinary complications showed that for 30-day and 90-day complications, troponin change was significant with OR 6.29 (95% CI 1.06 to 61.2) and 4.457 (95% CI 1.2 to 22.01), respectively, whereas for 6-month complications, the ACS NSQIP predicted morbidity was significant with an OR of 1.24 (95% CI 1.09 to 1.49).There was no difference in the incidence of urinary complications between the two groups (p 0.09) or day 30. Groupwise analysis of pulmonary complications showed no significant difference. Logistic regression showed that for day 180, the treatment group was the only significant factor, with the RIPC group having an OR of 0.2 (95% CI 0.03 to 0.92). No significant variables were identified for day 90 , 6.3 (95% CI 1.23 to 51.72) and 7.15 (95% CI 1.41 to 53.65) at day 30, 90 and 6 months, respectively. Further logistic regression with independent variables POMS gastrointestinal morbidity on days 2, 3 and 5 showed that only morbidity on day 5 was significant, with OR 6.48 (95% CI 1.48 to 33.77) at day 30, 5.963 (95% CI 1.35 to 31.28) at day 90 and 7.93 (95% CI 1.47 to 57.62) at day 180.p = 0.0006). Groupwise analysis showed that the difference was significant at all time points . Logistic regression showed that the treatment group was a significant independent variable, OR 0.04 , 0.12  and 0.12  at 30 days, 90 days and 6 months, respectively. Additionally, hs-TropI change was a significant predictor of 6-month complications, OR 7.14 (95% CI 1.16 to 408.54).Surgical complications developed in 4.5% vs. 33% at 30 days, 9.5% vs. 35% at 90 days and 11% vs. 41% at 6 months in the intervention vs. control group , which, in turn, was the only variable associated with prolonged length of stay. This finding is in line with previous studies that showed that POMS morbidity on postoperative days 3 and 5 after major surgery is associated with a longer hospital stay ,21.Amongst the surgical complications, the greatest difference between the two groups was observed in the incidence of anastomotic leak. No studies investigating the effect of RIPC on postoperative leak following bowel anastomosis were identified; however, a meta-analysis of gastric ischaemic preconditioning prior to oesophagectomy showed that the intervention reduces the incidence of severe anastomotic leak that requires reoperation  . Animal Regarding the postoperative pulmonary complications, the greatest difference between the two groups was noted in the incidence of pneumonia and respiratory failure. A study of RIPC in a rat cardiopulmonary bypass model showed that RIPC attenuated postoperative lung injury, as indicated by lower protein content in the bronchoalveolar lavage fluid, less severe alveolar wall thickening, reduced neutrophil infiltration and increased dynamic lung compliance. The anti-inflammatory cytokines interleukin-4 (IL-4) and IL-10 were found to be significantly higher in the serum of the RIPC compared to the control group, potentially indicating that the pulmonoprotective effects of RIPC are due to its anti-inflammatory effects .Perioperative hsTrop-I change in our study was an independent predictor of postoperative AKI, 30- and 90-day urinary complications and 6-month surgical complications. This outcome is in keeping with Noordzij’s study that showed that troponin rise following abdominal surgery, in addition to its association with increased mortality, also had an increased risk of non-cardiac complications such as sepsis, anastomotic leak, respiratory insufficiency, wound infection and bleeding .Our study adds further evidence on the use of RIPC in non-cardiac non-vascular surgery and is the first in our knowledge to study the effect of RIPC on TIMP-2*IGFBP-7 and POMS morbidity in this setting. One of its limitations is that, as a pilot study, it has not been powered to detect a difference between the intervention and control groups. Additionally, we assumed that the POMS on day 5 is zero for patients discharged prior to that day and our out-of-hospital assessment of morbidity has been cumulative over time, which, in combination with 30% of participants being lost at the 6-month follow up, may have overestimated the incidence of 6-month complications. Although the study aimed to recruit patients above a certain threshold of predicted morbidity undergoing abdominal surgery, each type of surgery is associated with its own specific potential complications and, given our relatively small sample size, our results should be interpreted with caution.In conclusion, our pilot study suggests that RIPC may limit postoperative morbidity in patients undergoing intra-abdominal cancer surgery and, given that RIPC is a simple, non-invasive and safe intervention, we recommend a definite suitably sized clinical trial.


Variable relative biological effectiveness (vRBE) in proton therapy might significantly modify the prediction of RBE-weighted dose delivered to a patient during proton therapy. In this study we will present a method to quantify the biological range extension of the proton beam, which results from the application of vRBE approach in RBE-weighted dose calculation.The treatment plans of 95 patients (brain and skull base patients) were used for RBE-weighted dose calculation with constant and the McNamara RBE model. For this purpose the Monte Carlo tool FRED was used. The RBE-weighted dose distributions were analysed using indices from dose-volume histograms. We used the volumes receiving at least 95% of the prescribed dose (V95) to estimate the biological range extension resulting from vRBE approach.The vRBE model shows higher median value of relative deposited dose and D95 in the planning target volume by around 1% for brain patients and 4% for skull base patients. The maximum doses in organs at risk calculated with vRBE was up to 14 Gy above dose limit. The mean biological range extension was greater than 0.4 cm.Our method of estimation of biological range extension is insensitive for dose inhomogeneities and can be easily used for different proton plans with intensity-modulated proton therapy (IMPT) optimization. Using volumes instead of dose profiles, which is the common method, is more universal. However it was tested only for IMPT plans on fields arranged around the tumor area.Adopting a vRBE model results in an increase in dose and an extension of the beam range, which is especially disadvantageous in cancers close to organs at risk. Our results support the need to re-optimization of proton treatment plans when considering vRBE.The online version contains supplementary material available at 10.1186/s13014-022-02022-5. Proton therapy (PT) is an effective technique for treating specific types of cancer, such as head and neck tumors . Thanks RBE) distributions. The latter is typically obtained, for protons, with a parametric model using the dose-averaged linear energy transfer (LETd) % (median 99.33%) for FRED cRBE model and% (median 99.80%) for FRED vRBE model.Two patient groups: brain patients (Patient 1–50) and skull base patients (Patient 51–95) were analysed separately. Patients were ranked according to increasing mean vRBE-weighted dose in the PTV. The comparison of relative mean dose in PTV to the prescribed dose are presented on the top panels of Figs. 95% of Dmean/Dp of median was% (median 98.44%) for FRED cRBE model and% (median 102.61%) for FRED vRBE model.For skull base patients the CIp value from Wilcoxon test performed between DRBE computed in FRED with cRBE and vRBE models was p < 0.05 , which means that the mean deposited dose in the PTV relative to prescribed doses in PTV (Dmean/Dp) are significantly different.The p = 0.002) as well as D05 values (p = 2.1 × 10–8), between DRBE distributions computed with the cRBE and vRBE models with CI95%  and . In the case of skull base patients the difference was also significant in both D95  and D05 .The values of D95 and D05 parameters computed for both groups of patients are presented in Figs. max) in brainstem and chiasm was analyzed for both groups of patients and presented on the fourth and the fifth panel of Figs. max in OARs was compared to the clinical constraints, which is 54 Gy(RBE) for brainstem core, 64 Gy(RBE) for brainstem surface and 54 Gy(RBE) for chiasm. The DVH analysis was performed on the whole brainstem structure. The median dose values computed with TPS and FRED for cRBE model are close to the dose constraints, while for the vRBE model the doses were much higher and often above dose constraints (up to 14 Gy above the limit), especially for skull base patients .The maximum dose , so even the slightly increased RBE can potentially impact the clinically relevant biological dose in brainstem or chiasm (with dose constraints for brainstem core and chiasm of 54 Gy(RBE)), and eventually have severe impact on these organs.In the PTV for brain patients (α/β = 6 Gy), the mean of the RBE values was 1.11, ranging from 1.07 to 1.31, whereas for skull base patients (α/β = 4 Gy) the mean RBE was 1.15 ranging from 1.09 to 1.49. In the brainstem the RBE values reached up to 2.71 and 2.36, while in chiasm the values were up to 2.16 and 2.19 for brain and skull base patients, respectively. The escalation of RBE values after the Bragg peak around 2.5 was also reported recently in Missiaggia et al.  and RvRBE was 4.70 cm with CI95% , whereas for skull base patients RcRBE was 3.00 cm with CI95%  and RvRBE 3.45 cm with CI95% . Results for all patients correlated with the volume of the V95 are presented on Fig. ext (formula(2)) for brain patients was Rext(brain) = 0.44(0.14) cm and for skull base patients Rext(skull base) = 0.45(0.11) cm.The mean RResults for all patients correlated with the volume of the V95 are presented on Figs. diffRobust was 0.7(1.0) Gy(RBE) and 3.2(2.6) Gy(RBE),CTVdiffRBE was 3.9(0.7) Gy(RBE) and 3.4(1.8) Gy(RBE)CTVThe results were analyzed and presented as DVHs of CTV (magenta curves), chiasm (green curves) and brainstem (blue curves) volume in Fig. iii.difRobust: 1.4(2.0) and 2.6(1.6) Gy(RBE), while OAR(chiasm)difRobust: 1.7(2.4) and 2.3(3.5) Gy(RBE),OAR(brainstem)iv.difRBE: 4.5(2.3) and 3.2(1.9) (RBE), while OAR(chiasm)difRBE: 4.3(2.3) and 3.4(2.3) Gy(RBE)OAR(brainstem)for brain and skull base patients, respectively.for brain and skull base patients, respectively. The mean differences for OARs form D05 parameter  were:iiResults for the selected brain and skull base patients show that incorporating the vRBE model can modify the shape of the DVH  compared to the cRBE model , to a lRBE. For brain and H&N patients Yepes et al. [The brain and skull base tumors are particularly challenging targets, because they are often localized very close to the critical structures . Minimizing the extension of the proton range and not exceeding dose limits in normal tissue is expected to be the factor limiting brain toxicity in H&N patients [By analyzing volumes receiving at least 95% of the prescribed dose using cRBE and vRBE we estimated the average biological range extension of over 0.4 cm. This result is similar to the predictions of biological range with vRBE obtained by comparing the edges of high dose regions (i.e. range at 80% of the prescribed dose) in Giovannini et al.  and Grünpatients .d and/or DRBE. As our results show, the MC simulations indicate that real dose distribution can be more inhomogeneous than predicted by the analytical TPS, which results in lower mean dose in PTV using the same RBE model, which was also reported in Tseng et al. [Monte Carlo simulation methods provide a reliable prediction of dose deposited in patients , 36. Dueg et al.  and Ytreg et al. . The RBEg et al. .d and variable RBE should support the treatment plan evaluation. Since including vRBE modeling in clinical treatment planning is still controversial, we believe that treatment planning optimization in the near future should include simultaneous optimization of LETd and biological dose with cRBE in critical structures [We suggest that MC calculations of LETructures . This wiRBE distribution, leading to an additional source of DRBE and range uncertainty in treatment planning. We suggest that the treatment planning procedure should account for the uncertainty of the DRBE, particularly in high dose regions, predicted with the vRBE models. The biological range extension due to vRBE can be even larger than planned margins in PTV. Our approach of estimating the biological range extension is a suitable method for IMPT plans because, differently to the analysis of transverse profiles, is insensitive to dose inhomogeneities and radiation field directions.As presented in this work, incorporating the vRBE approach would modify the DAdditional file 1: Table S1. Patient database grouped by tumor type with information on prescribed dose, PTV volume and diagnosis. For the skull base patients the reported volume is from the first stage of treatment.


Background: Effective treatment with tissue plasminogen activator (tPA) critically relies on rapid treatment. Door-to-needle time (DNT) is a key measure of hospital efficiency linked to patient outcomes. Numerous changes can reduce DNT, but they are difficult to trial and implement. Discrete-event simulation (DES) provides a way to model and determine the impact of process improvements.Methods: A conceptual framework was developed to illustrate the thrombolysis process; allowing for treatment processes to be replicated using a DES model developed in ARENA. Activity time duration distributions from three sites  were used. Five scenarios, three process changes, and two reductions in activity durations, were simulated and tested. Scenarios were tested individually and in combinations. The primary outcome measure is median DNT. The study goal is to determine the largest improvement in DNT at each site.Results: Administration of tPA in the imaging area resulted in the largest median DNT reduction for Site 1 and Site 2 for individual test scenarios . Ensuring that patients arriving via emergency medical services (EMS) remain on the EMS stretcher to imaging resulted in the largest median DNT improvement for Site 3 . Reducing both the treatment decision time and tPA preparation time by 35% resulted in a 11.0% (95% CI 10.0–12.0%) maximum reduction in median DNT. The lowest median and 90th percentile DNTs were achieved by combining all test scenarios, with a maximum reduction of 26.7% (95% CI 24.5–28.9%) and 17.1% (95% CI 12.5–21.7%), respectively.Conclusions: The detailed conceptual framework clarifies the intra-hospital logistics of the thrombolysis process. The most significant median DNT improvement at rural hospitals resulted from ensuring patients arriving via EMS remain on the EMS stretcher to imaging, while urban sites benefit more from administering tPA in the imaging area. Reducing the durations of activities on the critical path will provide further DNT improvements. Significant DNT improvements are achievable in urban and rural settings by combining process changes with reducing activity durations. Stroke is a devastating disease, but is treatable with alteplase or tissue plasminogen activator (tPA)  and endoSeveral process changes can reduce DNT , 14, butSeveral studies have illustrated the key activities involved in either the pre-hospital or intra-hospital aspects of acute stroke care –29, 33. A conceptual framework was developed first to provide a generalized model for tasks involved in the thrombolysis process within both urban and rural hospitals. This conceptual model was then used to develop a DES model that can be used for both urban and rural hospitals, based on various inputs. This paper includes descriptions of the conceptual framework, the DES model, as well as the test scenarios applied to the three included sites. The authors declare that all supporting data are available within the article and its Site Context section : Patients arriving via EMS remain on EMS stretcher to imaging : Administration of tPA in imaging area (regular and out of hours) (f hours) , 40–42—TProcess change 3 (P3): Pre-registration of patients arriving via EMS : Reduce treatment decision time by 35%—This scenario models a reduction in the time taken for the physician to decide whether the patient will receive thrombolysis treatment by 35%.Reduction in activity duration 2 (R2): Reduce tPA administration preparation time by 35%—This scenario models a reduction in the time taken to complete the preparation required before administering tPA by 35%. Tissue plasminogen activator administration preparation encompasses activities such as: obtaining the drug, estimating the patient's weight, calculating the correct dosing, mixing the tPA, and programming the tPA administration pump.th percentile DNT for patients treated with thrombolysis. This measure is important as it shows the DNT where most (90% of patients) are treated within, and there has been previous justification for the use of this measure in acute stroke processes . The reduction in IQR was also reported. A critical outcomes measure that was included was 90rocesses . The cure 60 min .p-value of < 0.05 was considered statistically significant.The Mann-Whitney U-test and the Chi-Square Goodness-of-Fit test were performed for continuous and categorical variables, respectively. Minitab Statistical Software for Windows  was used for all statistical analysis. A Section 2 Treatment Process Results section of the qualitative study , their arrival rates were adjusted accordingly. The time and day of a patient's arrival in the system can impact their treatment pathway due to resource availability. Regular hours are defined as 8:00 a.m. to 4:00 p.m., Mondays to Fridays, while out of hours are considered all times outside of regular hours. The schedule generates approximately 35% of arrivals in regular hours and 65% during out of hours as determined from our input data. The schedule specifies that the majority of arrivals occur between 8:00 a.m. and 8:00 p.m., with few arrivals between 12:00 a.m. and 8:00 a.m. Human resource and the resulting treatment process differences between regular and out of hours are defined for each site in the ve study . In addive study –6 and move study  can be fThe model activity duration distributions  and arriThe DES has several assumptions and simplifications:Due to the top prioritization of acute stroke patients within a hospital, it is assumed these patients will receive priority for the required resources upon arrival, and that the resources are always available.Patient pathway type assignments (EMS or PV) are independent.The EMS pathway always provides the hospital with pre-notification and pre-identification.Activity durations are independent of each other.All patients in the DES model are eligible to receive thrombolysis treatment.The system may have only a single stroke protocol activation at a time.The model assumes 100% stroke protocol compliance and does not consider personnel dependent variation. Although, it should be noted that waiting for a patient's lab results is not always required to determine the thrombolysis treatment decision, which is reflected in the duration distribution for this activity.There are various reasons based on literature for the established DES assumptions that allow the model to produce meaningful results. Assumption 1 was established due to the Canadian triage and acuity scale (CTAS) level assigned to AIS patients in the qualitative study which provided the foundation for this work . AssumptThe following are model simplifications:Activity durations are independent of the onset to arrival time, meaning durations do not change if a patient is approaching the 4.5-h window.Only AIS patients are included; stroke mimics, and hemorrhagic stroke patients are excluded.Ineligible thrombolysis candidates are not considered.Stroke severity, patient age, and patient sex are not considered.Certain variation in activities may be due to factors included in the assumptions and simplifications listed above; for example, milder stroke patients often have longer treatment times, due to the time taken to determine a diagnosis, and treatment decision. These variations in the associated activities are considered in the distribution of each relevant activity in the DES.The model did not include a warm-up period due to the transient nature of the treatment process being modeled. As randomness exists in all simulation results, a number of replications analysis was conducted to determine the number of runs required to achieve a particular precision of the outcome estimate, summarized in The simulation model was developed using ARENA software .To obtain access to the developed ARENA model, please contact the corresponding author.Ethics approval was obtained from the Nova Scotia Health Research Ethics Board (REB) for this study, with the REB file number 1025975.The model was used to determine the impact of process changes and reduction in activity durations on DNT at each site in comparison to the site-specific baseline DNT. The actual median DNT for Site 1, Site 2, and Site 3 are 50.0, 40.0 and 77.5 min (June 2019–May 2020), with modeled baseline median DNT of 50.0 (IQR 45.4–53.8), 40.1 (IQR 38.7–48.0), and 74.0 (IQR 70.8–82.6) min, respectively. The results of the model baselines and test scenarios are summarized in The results of all scenario experiments are described below and summarized in th percentile DNT results are summarized in Patients Arriving via EMS Remain on EMS Stretcher to Imaging (P1)—As Site 1 and Site 2 were currently implementing this process as their baseline, those sites did not experience an improvement in DNT. This process change did benefit Site 3, and led to the largest improvement for Site 3 across all individual test scenarios. The DNT at Site 3 was reduced to 66.4 (IQR 63.6–75.7) min from a median of 74.0 (IQR 70.8–82.6) min, translating to a 9.2% (95% CI 7.9–10.5%) improvement in DNT compared to the respective site baseline.Administration of tPA in Imaging Area (Regular and Out of Hours) (P2)—Administration of tPA to patients in the imaging area resulted in a reduction of the median DNT for Site 1, Site 2, and Site 3 to 43.7 (IQR 42.2–46.3) min, 36.6 (IQR 34.9–44.6) min, and 71.0 (IQR 66.0–80.3) min, respectively. This scenario was the most impactful individual test result for Site 1 and Site 2, leading to a reduction of 12.6% (95% CI 12.4–12.8%) and 8.2% (95% CI 7.5–9.0%), respectively. It was anticipated that Site 1 would show the largest improvement for this scenario, as Site 1 has the longest travel time to the ED from imaging since it is large tertiary care center.via EMS (P3)Pre-registration of Patients Arriving —As Site 1 and Site 2 include this process in their baseline, those sites did not experience an improvement in DNT. This process change resulted in a DNT of 71.9 (IQR 68.6–80.9) min and a reduction of 1.7% (95% CI 0.3–3.1%) at Site 3.Reduce Treatment Decision Time by 35% (R1)—Physician comfort with treatment with tPA results in a shorter amount of time for making the treatment decision. The reduction in decision time did not reduce Site 1's DNT, highlighting that a bottleneck was present elsewhere. Scenario R1 also had little impact on Site 2, with a 0.5% (95% CI −0.1–1.2%) improvement in DNT. Site 3 experienced a 1.8% (95% CI 1.0–2.7%) improvement, with a DNT of 72.3 (IQR 70.0–76.0) min.Reduce tPA Administration Preparation Time by 35% (R2)—This reduction in activity duration scenario benefitted Site 1 and Site 2 to a greater degree than did R1. The DNT was reduced for Site 1 by 8.6% (95% CI 8.2–9.0%) with a DNT of 45.7 (IQR 42.0–49.1) min. Site 2 improved their DNT by 5.3% (95% CI 4.2–6.5%) with an output of 37.8 (IQR 35.9–48.0) min. The impact of R2 was comparable to R1 for Site 3, with DNT reduction of 1.6% (95% CI −0.2–3.5%) and a DNT of 71.8 (IQR 66.6–81.4) min.Combination of P1, P2, P3—When combining all process change scenarios, Site 3 saw a 15.4% (95% CI 13.1–17.7%) reduction with a DNT of 61.3 (IQR 56.3–72.6) min from a baseline median of 74.0 (IQR 70.8–82.6) min. As Site 1 and Site 2 were currently implementing P1 and P3, their respective results are identical to their P2 results as that was the only new process change for those sites.Combination of R1, R2—Combining the two reductions of activity duration scenarios benefited Site 3 far more than Site 1 and Site 2, whose improvements in this case were largely attributable to R2. The combination of R1 and R2 was equally, or more, effective in terms of improving DNT when compared to being individually tested, due to the close relationship of these activities and the bottleneck formed when adjusted individually. The resulting DNT for Site 1, Site 2, and Site 3 were 45.7 (IQR 42.0– 49.1) min, 36.7 (IQR 35.4–48.0) min, and 65.3 (IQR 62.8–70.6) min, respectively.Combination of P1, P2, P3, R1, R2—As anticipated, the lowest DNT for each site was achieved by combining all test scenarios, which resulted in the following DNTs: Site 1 was reduced by 18.9% (95% CI 18.6–19.1%) to 40.6 (IQR 39.1–43.2) min, Site 2 by 15.9% (95% CI 15.3–16.4%) to 33.6 (IQR 32.4–44.6) min, and Site 3 by 26.7% (95% CI 24.5–28.9) to 52.9 (IQR 49.8–61.2) min.th percentile DNT reduction percentages results which further define the spread of the DNT results. As shown in th percentile DNT experienced in individual test scenarios for Site 1, Site 2 and Site 3, respectively, as shown in th percentile DNT results when considering all individual and combinations of test scenarios were achieved by combining all three process changes and two reduction in activity durations for all sites. When combining all test scenarios, the following 90th percentile DNT improvements were experienced: Site 1 was reduced from 71.3 to 63.0 min , Site 2 was reduced from 62.8 to 56.2 min , and Site 3 was reduced from 92.9 to 82.7 min .In addition to median DNT and DNT reduction percentages results, The developed conceptual framework expands upon published works  to incluth percentile DNT  modelers must understand clinical concepts and have the ability to grasp the pathway logistics; and (2) translate that knowledge into a model for use of testing . This hiA limitation of this study is that the conceptual framework was based on only three hospitals with a small sample size of participants involved at each site in a single Canadian province . Furtherth percentile DNT in urban and rural settings. Our modeling shows that of the three included process changes, the most significant median DNT improvement at rural hospitals resulted from ensuring patients arriving via EMS remain on the EMS stretcher to imaging, while urban sites benefit greater from administering tPA in the imaging area. Reducing the durations of activities found on the critical path will result in further DNT improvements. The lowest median DNTs and 90th percentile DNTs results were achieved by combining all three process changes and two reduction in activity durations for all sites. Significant DNT improvements are achievable in urban and rural settings by combining process changes and reducing activity durations. Based on the results of this study, similar sites can feel confident in achieving desired outcomes when implementing these process improvements. Discrete-event simulation modeling of acute stroke processes can be expanded to develop more accurate models to reveal the ideal processes to achieve the most efficient treatment for patients.The conceptual framework developed in this study defines treatment pathways for AIS patients receiving thrombolysis treatment in urban or rural hospitals. The detail of the framework aims to reduce the gap between clinical knowledge and modeler perceptions regarding the logistics of the intra-hospital aspect of the thrombolysis process. Several process improvements were shown to have a positive impact on median DNT and 90The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.TB: study design, DES modeling, data analysis, preparation of figures and tables, preparation of the first and revised drafts of the manuscript, as well as final editing, and formatting. DV: revision of manuscript for intellectual content. JB: input into the following areas: data analysis, model verification, sensitivity analysis, statistical analysis, as well as revision of manuscript for intellectual content. NK: study design, input into data analysis, editing, formatting, and revision of manuscript for intellectual content. All authors contributed to the article and approved the submitted version.This work was funded by Canadian Institutes of Health Research (CIHR) Project Grant (PJT-169124) held by NK.NK: Founder and part equity owner of DESTINE Health. Principal Investigator for CIHR grant that funded this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To assess changes in ankylosing spondylitis (AS) incidence, prevalence and time to diagnosis, between 1998 and 2017.Using UK GP data from the Clinical Practice Research Datalink, we identified patients diagnosed with AS between 1998 and 2017. We estimated the annual AS incidence, prevalence and length of time from first recorded symptom of back pain to rheumatology referral and diagnosis.We identified 12 333 patients with AS. The incidence declined from 0.72 (±0.14) per 10 000 patient-years in 1998 to 0.39 (±0.06) in 2007, with this decline significant only in men, then incidence rose to 0.57 (±0.11) in 2017. By contrast, prevalence increased between 1998 and 2017 (from 0.13%±0.006 to 0.18%±0.006), rising steeply among women (from 0.06%±0.05 to 0.10%±0.06) and patients aged ≥60 (from 0.14%±0.01 to 0.26%±0.01). The overall median time from first symptom to rheumatology referral was 4.87 years (IQR=1.42–10.23). The median time from first symptom to diagnosis rose between 1998 and 2017 (from 3.62 years (IQR=1.14–7.07) to 8.31 (IQR=3.77–15.89)) and was longer in women (6.71 (IQR=2.30–12.36)) than men (5.65 (IQR=1.66–11.20)).AS incidence declined significantly between 1998 and 2007, with an increase between 2007 and 2017 that may be explained by an improvement in the recognition of AS or confidence in diagnosing AS over time, stemming from increased awareness of inflammatory back pain and the importance of early treatment. The rising AS prevalence may indicate improved patient survival. The persisting delay in rheumatology referral and diagnosis remains of concern, particularly in women. Data on the incidence and prevalence of ankylosing spondylitis (AS) are sparse.There is a significant diagnostic delay in AS, which results in worse patient outcomes.This population-based study, conducted in the UK across two decades and including 12 333 patients diagnosed with AS, showed a decrease in the incidence of AS among men and a rise in prevalence, especially in the over 60s.The observed lack of improvement in diagnostic delay over a 20-year period, appeared to be largely driven by delay in referral from primary care to rheumatology.These results highlight the need to promote the recognition of inflammatory back pain among all health practitioners evaluating people with back pain, and to encourage referral of suspected AS cases to rheumatology.Ankylosing spondylitis (AS) is a disabling chronic arthritis characterised by systemic inflammation leading to fusion of the axial skeleton, and represents the established phenotype within the axial spondyloarthritis (axSpA) disease spectrum. AS typically presents in a patient’s mid-20s and more commonly in men,3 4Estimates of the incidence and prevalence of AS range from 0.05 to 1.4/10 000 person-years and from 0.1% to 1.4%, respectively.6Treatment initiation in the earlier stages of disease associates with better clinical response and increased likelihood of remission.Another important factor is the absence of diagnostic criteria for AS, despite the existence of diagnostic algorithms for axSpA.Using a large UK primary care dataset, we investigated trends in the incidence, prevalence and time to rheumatology referral and diagnosis in AS over two decades, to assess disease burden and the impact of modern practices in the diagnostic approach.This retrospective study is reported following current guidelines for observational studies  GOLD dataset, which contains 17.6 million electronic health records from 734 UK general practitioner (GP) practices (approximately 8% of UK GP practices). The data source was established in 1987 and contains records that span the duration of a patient’s lifetime.The eligible population had ≥1 day of registration during the study period (1 January 1998 to 1 April 2018) and excluded records flagged with ‘unacceptable’ research quality. Patient follow-up commenced from the latest of: the study start date, being aged ≥18 years, and having 1 year of UTS practice registration.The Read V.2 code, ‘N100.’ ‘Ankylosing spondylitis’, was used to define AS diagnoses and we excluded patients diagnosed before age 18 years. In a sensitivity analysis, we required an additional diagnostic or AS measurement code >7 days later, to improve diagnostic certainty . The AS The outcomes were the annual AS incidence and prevalence by sex, age and geographical area, and the annual time to diagnosis among women and men. Time to diagnosis was defined as the number of years between first coded non-specific back pain symptom and AS diagnosis.We reported baseline patient characteristics and annual trends in outcomes (1 January 1998 to 31 December 2017). The sensitivity analysis ran until 31 December 2016 to enable >16 months of follow-up for the additional AS coding. The difference between calendar years was deemed significant where the 95% CIs did not overlap. All analyses were stratified by sex, except for years with ≤5 women or men.Crude annual and period incidence rates per 10 000 person-years were calculated with 95% CIs for patients ‘at-risk’, that is having no AS diagnosis and ≥1 year of prior GP registration at the start of that time-period.The earliest recorded back pain symptom, and the first subsequent rheumatology referral, prior to AS diagnosis were identified. The median times between these were calculated overall and for patients diagnosed with AS in each year. Patients with ≥2 and ≥3 years of quality (UTS) registration prior to AS diagnosis were included in subanalyses of time from symptom to diagnosis.R V.3.6.2, Microsoft SQL 2017 and Microsoft Excel 2016 were used.A cohort of 12 333 patients with AS was identified (excluding 302 patients diagnosed aged ≤18), of whom 26.0% (N=3209) were women . The medThere were 3101 patients in the incident AS cohort , of whom 30.8% (N=951) were women. In the incident cohort, the median age at diagnosis was 43 (IQR=33–56) and the median duration of follow-up per patient was 12.6 (7.7–16.1) years. In sensitivity analyses (having a subsequent diagnostic or measurement code >7 days later), there were 1071 incident patients with AS, of whom 27.9% (N=299) were women and the median age at diagnosis was 40 (IQR=32–51) years.The period incidence was 0.54 (±0.02) per 10 000 person-years; 0.19 (±0.01) in sensitivity analyses . The at-Incidence declined significantly from 0.72 (±0.14) in 1998 to 0.39 (±0.06) in 2007, then rose significantly to 0.57 (±0.11) in 2017 . The meaIn sensitivity analyses, incidence was 0.34 (±0.09) in 1998 and declined more steeply to 0.13 (±0.04) in 2007, but then stabilised (0.10±0.04 in 2016). The mean APC was −2.91: −9.89 in 1998–2002 and +1.07 in 2012–2016. The initial decline was significant in both sexes, with no significant change after 2007 . IncidenThe period prevalence of AS was 0.15% (±0.003) . PrevalePrevalence rose significantly from 0.13% (±0.01) in 1997 to 0.18% (±0.01) in 2017; more steeply in women than men (mean APC +2.69 and +1.07) . Annual The annual prevalence was stable in sensitivity analyses (0.07% ±0.004 in 1998 and 2016), although there was a rising trend in women (mean APC +1.43). The annual prevalence declined in patients aged <60, and rose in older cohorts, notably in patients aged 70–79 . There wAt AS diagnosis, 2120 (68.4%) patients had a prior-recorded back-pain symptom. The proportion with a prior-recorded back-pain symptom rose between 1998 and 2017 (from 60.4% to 77.6%) and was higher in women (70.6%) than in men (67.4%) . ResultsThe median time from first back pain symptom to diagnosis was 6.0 years (IQR=1.9–11.6), higher in women  than men  and in subanalyses with ≥2 and ≥3 quality registration years . In sensitivity analyses, the time to diagnosis was lower , again higher in women  than men  and higher in UTS-related subanalyses .During the study period, time to diagnosis more than doubled from 3.6 (IQR=1.1–7.1) years  in 1998 to 8.3 (3.7–15.9) years  in 2017 . In sensThe recording of rheumatology referrals increased over time . The medIn this first study of AS incidence and prevalence in the UK, incidence appeared to decline between 1998 and 2007. From 2007 to 2017, there was an apparent rise in incidence that was not seen in the sensitivity analysis. Prevalence rose among patients aged ≥60 and among women. There was a trend to worsening in delay to referral and diagnosis over two decades, particularly in women, with no reduction in the sex difference.From 2007 to 2017, an increasing proportion of patients had a single (rather than ≥2 instances of) AS code during follow-up . This may suggest increased awareness of AS, in cases where it indicates that AS was suspected but not confirmed. British guidelines published in 2005 on treating AS with biologicals may have raised awareness and prompted investigation of suspected cases of AS.The period prevalence in our study was comparable to the weighted mean prevalence of 0.19% estimated from other European countries.25It is important to note that we found no improvement in the diagnostic delay of AS since this was first highlighted in 1999,32A positive finding, however, is that the recording of back pain symptoms increased over time. This suggests that while a survey published in 2011 found that almost half of patients with AS waited over a year after developing symptoms before contacting a healthcare professional,The increase in the recording of referrals over time suggests increasing awareness among GPs of AS symptoms and the importance of referral for rheumatology-led biological therapy.10 30Study strengths included long-term follow-up (median 12.6 years) of a large population-based cohort, which is important for the identification of early symptoms and rheumatology referrals recorded in the years prior to the diagnosis of AS. We explored AS in primary care where symptoms first present, potentially capturing early presentations of back pain symptoms and ensuring that GP-diagnosed cases were not missed.et al reported that, over time in the UK, diagnoses of AS have increasingly been made in the specialist rheumatology set up rather than primary care.Study limitations include those common to routine data-based studies, for example, incomplete and changing coding practices.As characterises open cohort observational studies, we used a dataset with a changing population structure and varying follow-up duration. However, we designed our investigation to account for this, including by reporting incidence with the denominator in person-years from an at-risk cohort and point prevalence rather than annual period prevalence.36In conclusion, the results of the primary and sensitivity analysis suggest that between 2007 and 2017 the incidence of AS may have stabilised or increased and awareness may have improved among GPs in the UK. The rising prevalence in the over 60s may indicate improved survival and highlights the importance of appropriate AS management in an ageing population. Unfortunately, the delay to diagnosis particularly in women has persisted over 20 years and appears to be largely driven by delay in referral to rheumatology. This delay highlights the need for education and increased awareness of IBP and associated AS features among non-rheumatologists.


To explore the relationship between retinal fluid status and best-corrected visual acuity (BCVA) in patients treated with intravitreal aflibercept (IVT-AFL) treat-and-extend (T&E) in the ALTAIR study.Outcomes were investigated according to overall fluid status at week 16 (predefined) and the relationship between any fluid, intraretinal fluid (IRF), subretinal fluid (SRF), or pigment epithelial detachment with BCVA at baseline, and weeks 16, 52, and 96 (post-hoc). The analyses involved treatment-naïve patients (N = 246) with exudative age-related macular degeneration (AMD), aged ≥ 50 years with BCVA of 73–25 Early Treatment Diabetic Retinopathy Study letters, who participated in the ALTAIR study.The mean (standard deviation) change in BCVA from baseline to week 52 was + 10.6 (10.9) and + 6.5 (16.0) letters in patients without and with fluid at week 16, respectively; and to week 96 was + 9.1 (14.3) and + 4.3 (16.1) letters in patients without and with fluid at week 16, respectively. The last injection interval was 16 weeks in 33.6% and 2.0% (week 52), and 62.9% and 17.6% (week 96) of patients without or with fluid at week 16, respectively. At baseline, 35.7% of patients had IRF and 85.2% of patients had SRF, which decreased to 11.8% (IRF) and 31.7% (SRF) of patients, 8.5% (IRF) and 18.7% (SRF), and 6.5% (IRF) and 20.7% (SRF) at weeks 16, 52, and 96, respectively. Presence of IRF at all timepoints was associated with poorer BCVA than if IRF was absent, while the presence of SRF was not associated with poorer BCVA compared with the absence of SRF.IVT-AFL T&E dosing was effective at clearing fluid regardless of fluid type in ~ 80% of patients with exudative AMD. Patients without fluid at week 16 had numerically better BCVA than those with fluid at week 16. Over 60% of patients without fluid at week 16 achieved the maximum treatment interval of 16 weeks by study end, compared with < 20% of patients with fluid at week 16. IRF , although evident in a small number of patients, was associated with poorer BCVA, whereas SRF was not.ClinicalTrials.gov Identifier: NCT02305238 Exudative age-related macular degeneration (AMD) is characterized by abnormal growth of new blood vessels in the macula  and is tTreat-and-extend (T&E) is a proactive, individualized dosing strategy whereby the treatment interval is gradually extended or shortened based on physician decision regarding the maintenance of functional and anatomic stability . The fleIncreasing evidence suggests that consideration of fluid, and the role of the different compartments, may be important in determining the impact of anti-VEGF treatment on functional outcomes in patients with exudative AMD –8. An anThis article reports predefined and post-hoc exploratory analyses of data from ALTAIR, a 96-week, randomized, open-label, phase 4 study  that was conducted to investigate the efficacy and safety of repeated doses of IVT-AFL with two different T&E approaches in patients with exudative AMD ) of 9.4 (1.7) and 11.8 (2.8) injections, respectively Table  and 17.6At baseline, mean (SD) BCVA was 55.5 12.4) and 54.5 (12.8) letters for patients without and with fluid at week 16, respectively. For patients without fluid at week 16, the mean (SD) change in BCVA from baseline to week 52 was + 10.6 (10.9) letters, and to week 96 was + 9.1 (14.3) letters. For patients with fluid at week 16, the mean change in BCVA was + 6.5 (16.0) letters and + 4.3 (16.1) letters from baseline to weeks 52 and 96, respectively  CRT was 370.8 (137.9) µm and 390.0 (145.1) µm for patients without and with fluid at week 16, respectively. For patients without fluid at week 16, the mean (SD) change in CRT from baseline to week 52 was − 132.0 (135.3), and to week 96 was − 134.5 (133.8) µm. For patients with fluid at week 16, the mean change in CRT was − 127.8 (138.9) and − 118.9 (144.4) µm from baseline to weeks 52 and 96, respectively , or who did not have a subretinal hemorrhage were more likely to have retinal fluid at week 16 Table . Mean  was 57.1 at baseline (n = 16), 66.1 at week 16 (n = 149), 65.4 at week 52 (n = 181), and 64.1 at week 96 (n = 179) for patients without any fluid Fig. .1 The prSRF was not associated with lower BCVA values, unless IRF was also present . Overall, 33 patients had new and/or persistent SRF both at weeks 16 and 52. Of these, 30 patients did not have coexisting IRF at week 52, and, in this group, the mean BCVA was maintained from week 16  to week 96 . Of the 33 patients with new and/or persistent SRF both at weeks 16 and 52, 3 patients had coexisting IRF at week 52, and, in this group, the mean BCVA decreased from week 16  to week 96 .In this predefined fluid analysis of data from the ALTAIR study, IVT-AFL T&E dosing was effective at clearing fluid and improving vision by week 16 in treatment-naïve patients with exudative AMD. The mean BCVA was similar between groups at baseline, and the mean change to week 52 was + 10.6 letters and + 6.5 letters in patients without and with fluid at week 16, respectively, with outcomes maintained to week 96. The mean change in CRT from baseline to week 96 followed a similar pattern; however, at baseline, there was a slight difference between groups . Regarding the last treatment interval up to week 96, over 60% of patients without fluid at week 16 achieved the maximum treatment extension interval of 16 weeks, compared with 17.6% of patients with fluid at week 16. From baseline to week 96, patients without and with fluid at week 16 received a mean of 9.4 and 11.8 injections of IVT-AFL, respectively. The post-hoc evaluation of baseline features suggested that patients with relatively thinner CRT and a lower PED height at baseline were more likely to have no fluid at week 16. A higher proportion of patients without fluid at week 16 had PCV and subretinal hemorrhage at baseline than those with fluid at week 16. Previous studies involving patients with PCV have reported comparable visual outcomes to those with typical AMD following treatment with IVT-AFL fixed or T&E dosing [Overall, the fluid compartment analysis showed that IVT-AFL T&E dosing was effective at clearing fluid in ~ 80% of patients with exudative AMD over the 96-week treatment period and led to improved BCVA, regardless of the fluid type. Over 70% of patients had no new or persistent IRF or SRF at any measured timepoint after week 8, following the three initial doses of IVT-AFL. The role of the different fluid compartments in determining the impact of anti-VEGF treatment on functional outcomes in patients with exudative AMD is a growing area of interest . IRF accIn our analysis, conducted in patients treated with IVT-AFL, new or persistent SRF  was not associated with lower BCVA, consistent with previous reports –8. HowevThe present analysis explored the relationship between fluid status at week 16 after initial dosing and BCVA following treatment with IVT-AFL in a T&E regimen over 2 years, suggesting that absence of fluid at week 16 was predictive of good outcomes. In the ALTAIR study , patientThe main strength of this study was that the data were obtained from a large randomized trial with a strict protocol, offering the opportunity to explore the relationship between fluid status and BCVA/anatomic outcomes in patients with exudative AMD. Furthermore, to our knowledge, this was the first CATT-like fluid analysis in patients treated with IVT-AFL. We should acknowledge that the CATT analysis identified various other factors  as beinA limitation of these analyses was the differing definitions of fluid between the predefined analysis, which was based on the presence or absence of new or persistent fluid, and the post-hoc analysis, which was based on the presence or absence of any fluid . Furthermore, the original study was not designed to determine the relationships between compartment fluid status and impact of disease outcomes after IVT-AFL treatment, and as the specific volume of fluid was not evaluated, quantitative fluid analysis could not be conducted. Limitations in relation to SD-OCT techniques and SD-OCT scan analysis for the determination of fluid status must also be acknowledged. Although there was no reading center involved in the ALTAIR study, SD-OCT machine and image settings for OCT were the same throughout the study at each site. The SD-OCT images were not read by the same observer nor were they checked by another individual, but were guided by a specific OCT manual.In conclusion, this analysis of data from the ALTAIR study demonstrated that IVT-AFL T&E dosing was effective at clearing fluid in over 70% of treatment-naïve patients with exudative AMD and improving vision. Findings from the predefined analysis demonstrated that patients without fluid had better BCVA than those with fluid at week 16. In patients with exudative AMD, baseline features such as CRT and PED height may play a role in determining the initial response of retinal fluid to treatment with IVT-AFL; further research is required to investigate predictive factors for response. The post-hoc analysis showed that IRF, although evident in a small number of patients, was associated with lower BCVA, whereas foveal SRF was not. Further quantitative analyses are required to confirm these findings.