To study for the first-time, pain perception, pain sensitivity, and self-reported pain in young adults with long disease duration of juvenile idiopathic arthritis (JIA) compared with controls.Children from Central Norway diagnosed with JIA between 1997 and 2004 were included consecutively in a population-based prospective study. Children with onset 1997–2000 were part of the Nordic JIA cohort. Controls were age- and sex-matched. In 2015–2017, study visits with investigator-blinded quantitative sensory testing (QST) comprising cold and warm detection thresholds (CDT/WDT), cold and heat pain thresholds (CPT/HPT), pressure pain threshold (PPT), and a suprathreshold heat pain test were performed. We constructed separate multilevel models for each variable of detection and pain thresholds with interaction between groups and site adjusted for the effect of age and sex.Among 96 young adults with JIA, 71% were female, median age was 22.7 years, disease duration was 16.1 years, and 47% had oligoarticular disease. Among 109 controls, 71% were female, and median age was 23.5 years. Participants with JIA had lower pressure pain thresholds (PPTs) (95% CI) compared to controls, upper limb 888  versus 1029  kPa and lower limb 702  versus 760  kPa. Participants with inactive disease had the lowest PPTs and cold pain thresholds (CPTs), compared to those in remission off medication and those with active disease. Minor differences were found regarding CDT/WDT and CPT/HPT in JIA compared to controls. The median (IQR) temperature needed to evoke pain = 6 on a 0–10 numeric rating scale (NRS) in the suprathreshold heat pain tests were lower in JIA than in controls (46 °C (45–47 °C) versus 47 °C (46–48 °C)). We found no associations between self-reported pain and pain thresholds.Our results indicate for the first time that young adults with long disease duration of JIA may have altered pain perception and sensitivity compared to controls. A clinical implication may be the importance of early treatment to quickly achieve pain-free remission and avoid long-term pain sensitization. Juvenile idiopathic arthritis (JIA) is the most common of the pediatric rheumatic diseases, affecting 12.8–23/100,000 children in the Nordic countries  with a wPain is the most frequent complaint in JIA and has a major negative effect on health-related quality of life (HRQoL), despite modern treatment and good disease control –10. The It has been a concern that neurobiological changes caused by prolonged exposure to pain early in the disease course of JIA, may induce a permanently altered pain perception and sensitization in the central nervous system , 14. TheDespite very few studies on pain thresholds in JIA, lower pain thresholds have been demonstrated, both in active and inactive disease , 21–23. The general aim of this study was to examine pain sensitivity and sensory perception among young adults with JIA included in a long-term follow-up study. The primary objective was to study whether pressure pain threshold, cold and heat pain thresholds and suprathreshold pain perception differed compared to matched controls. A secondary objective was to explore potential associations between self-reported pain and pain thresholds.Consecutive, newly diagnosed children with juvenile idiopathic arthritis (JIA) from Central Norway, with disease onset between January 1, 1997, and November 30, 2004, were prospectively included in this population-based longitudinal study. Children with onset between January 1, 1997, and June 30, 2000, were part of the 18 years follow-up of the multicenter cohort study, the Nordic JIA study , 27. To Participants ≥ 16 years with at least a baseline visit, scheduled to take place 6 months after disease onset, and a follow-up visit between 2015 and 2017 with a quantitative sensory test (QST), were included.At the baseline visit, demographic and clinical examination, medication, disease activity, and blood tests were registered, and participants or their parents depending of the participant’s age, filled in self-reported questionnaires. After the baseline visit, participants had yearly follow-up visits until they became 18 years and again at the present follow-up. The present study included a visit with clinical examination, blood tests, an updated registration of disease status, and previous and ongoing medication. Self-reported health-related quality of life (HRQoL) questionnaires, including pain measurements, were filled in.From the National Population Register of Norway, age- and sex- matched controls were randomly selected from rural and urban areas of Central Norway. Letters were sent to eligible controls inviting them to participate if they had no rheumatic disease, cancer, or autoimmune diseases. Reminder letters and Short Message Service (SMS) were sent if no answer.Clinical inactive disease was defined according to the American College of Rheumatology (ACR) provisional criteria including; no active joints, no fever, rash, serositis, splenomegaly or generalized lymphadenopathy attributable to JIA, no active uveitis, normal erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP), physician’s global assessment of disease activity with best attainable score indicating no disease activity, and patient-reported morning stiffness ≤ 15 min . AccordiTo evaluate HRQoL among JIA and controls we used the Medical Outcomes Study 36-Items Short-Form Health Survey (SF-36), a generic multidimensional measurement giving a physical and mental component summary (PCS/MCS) score  . Based oImmediately before the experimental test procedure both participants with JIA and controls registered current joint-related pain on a 10-cm VAS scale  and filled in a questionnaire, including questions on medication, food, caffeine, alcohol, and use of tobacco on the day of visit. Female participants registered last menstrual period and hormonal contraceptive use.All tests were performed in one experimental session by the same experienced investigator, who was blinded to whether the participant was a person with JIA or a control. Identical instructions were given by the investigator. The experimental procedure was performed in a constant order and was identical to the protocol used by Iversen et al. . ParticiTo measure thermal thresholds, we used the Somedic MSA thermal stimulator  with a hand-held Peltier element thermode with contact area of 25 × 50 mm . Pre-defined cut-off temperatures were 5 and 52 °C. To determine thermal thresholds we used the method of limits . The act2 rubber tip fixed to the gauge. Two sites were used; the medial phalanx of the third finger on the left hand, and the right leg, 16 cm distal to the inferior patella margin of the medial anterior tibia. Pressure was applied manually with an even rate perpendicular to the site investigated. Participants were instructed to verbally communicate whenever the sensation of pressure changed into the first sensation of pain, and the application of pressure stopped immediately. PPT was measured 3 times at each site [To estimate the pressure pain threshold (PPT), we used a hand-held digital pressure algometer  with a 1-cmach site , 36.To investigate the suprathreshold heat pain response, we used the Somedic MSA thermal stimulator, controlled by the Exposure30 software. Suprathreshold heat pain responses were obtained with continuous stimulation of heat pain on the left volar forearm. The temperature was individually determined corresponding to verbal numeric rating scale (NRS) = 6, ranging from 0 (no pain) to 10 (unbearable pain). At least 2 subsequent 7-s stimuli of heat, with 1-min intervals were used to select the temperature corresponding as close as possible to NRS = 6.At the actual suprathreshold heat pain procedure, a continuous stimulation of 120 s at the predetermined temperature was performed. Participants were informed to verbally report their NRS pain scores whenever it changed, not being informed that the temperature was held constant. The last NRS value reported every 10 s from 10 to 120 s, were recorded. If the participants terminated the procedure, a new sequence was carried out with 1 °C lower temperature. The suprathreshold heat pain procedure was based on the protocol by Uglem et al. , originaU test or Student’s t test. Raw data of cold pain threshold (CPT), heat pain threshold (HPT), and pressure pain threshold (PPT) were presented in median and dot-plots. We constructed separate multilevel models for each variable of detection and pain thresholds with interaction between groups and site adjusted for the effect of age and sex. The models were specified as three-level models with subject as third level random effect and site as second level. Analysis of CPT was done by a multilevel Tobit-model to account for censored values (thresholds below the 5 °C floor temperature). Post hoc contrasts of estimated margins were used for ANOVA-style tests of main, interaction, and simple effects. The results were presented as differences in degrees from baseline (32 °C) in cold and warm detection threshold (CDT/WDT), absolute temperatures in CPT/HPT, and as kilopascal in PPT with 95% CI accompanied by p values.To summarize clinical characteristics, we used descriptive statistics with absolute frequencies and percentages for categorial variables, and median with 1st–3rd interquartile range (IQR) or mean with standard deviations (SD) for continuous variables. To investigate group differences in pre-test characteristics, we used Mann-Whitney To investigate association of subgroups of JIA with pain thresholds, we constructed additional multilevel models for each variable of the pain thresholds . To evaluate potential associations of self-reported pain with pain threshold values, self-reported pain was included as predictor variable in these models.To evaluate the model, the residuals were plotted on histograms and quantile-quantile plots and the distributions for pain thresholds were found acceptable. Detection thresholds were transformed logarithmically to optimize the distribution of residuals. A robust estimator of variance was applied for HPT and PPT.t test.For analyses of the suprathreshold heat pain test, we used a multilevel model with interaction between time and groups, specified as a two-level model with subject as random intercept and time as random slope with an unstructured covariance matrix. Differences in temperature equaling numeric rating scale (NRS) = 6 was explored with Student’s Given 100 participants with JIA and 100 controls, the power was calculated to be 94% to detect a difference corresponding to 0.5 SD and 80% to detect a difference of 0.4 SD in the mean value of a continuous variable between the two groups. For dichotomous variables, the power was calculated close to 80% to detect a difference in proportions between the groups corresponding to 0.5 in JIA and 0.3 in controls.All statistical analyses were performed using STATA version 16, software .In the present study 96 of 114 (84%) eligible participants with JIA completed a clinical visit and a quantitative sensory testing (QST). At the study visit, median age of participants with JIA was 22.7 years, 71% were female, and median disease duration was 16.1 years (IQR 14.2–17.1) (Table p = 0.004). In the JIA group 16% reported poor health (SF-36 PCS < 40) compared to 7% among controls (p = 0.06), and 22% reported poor mental health (SF-36 MCS < 40) compared to 17% among controls (p = 0.3). We did not find any group differences in background characteristics regarding alcohol, tobacco, psychotropic, or pain medication on the day of visit.Participants with JIA reported lower mean SF-36 PCS compared to controls (51.1 (± 9.5) versus 55.1 (± 7.9), The results of the detection thresholds are shown in Supplementary Table SMedian cold pain threshold (CPT) was slightly lower (higher absolute temperature) in the JIA group compared to controls Fig. . Due to The upper limb was more sensitive to thermal stimuli than the lower limb for both JIA and controls, except for CPT in individuals with inactive JIA. Females and younger participants were more sensitive to thermal stimuli compared to males and older participants. Only minor differences between JIA categories were found, but the numbers in some categories were small Table .p < 0.001) and 702  versus 760  kPa for lower limb (p = 0.01) (Table Median pressure pain threshold (PPT) was lower among participants with JIA compared to controls both at upper and lower limb Fig. . Results02 670, 74 versus p = 0.02). During the continuous stimulation of heat pain for 120 s, NRS increased from 5.84 to 6.80 in the JIA group and from 6.00 to 6.59 in the control group . Two participants (one from each group) did not obtain a complete test and were excluded from the analyses.The median IQR 1st–3rd) temperature equaling numeric rating scale (NRS) = 6, differed between individuals with JIA and controls (46 °C (45–47 °C) versus 47 °C (46–48 °C), rd temperp < 0.001, respectively  VAS pain 1.0 (± 1.7) versus 0.2 (± 1.0), ly Table . A similWe did not find any association between self-reported pain scores, and the quantitative sensory testing (QST) results, neither among the JIA group, nor among controls , and slightly lower median cold pain threshold (CPT) (higher absolute temperature) compared to controls. Participants with JIA had lower pressure pain threshold (PPT) compared to controls, and participants with inactive JIA had the lowest thresholds. Results from the suprathreshold heat pain test showed that participants with JIA reported lower temperature equaling numeric rating scale (NRS) = 6 compared to controls and tended to have a steeper rise in the NRS score. Self-reported pain scores were higher in the JIA cohort compared to controls, and participants with active JIA had the highest pain scores. No associations between self-reported pain and pain thresholds were found.The main strengths of our study are the population-based design and long and equal follow-up time for all JIA participants, which enhances the generalizability of the findings and allows for evaluation after long disease duration, even though the quantitative sensory testing (QST) results are cross-sectional. Another strength is the standardized experimental procedure using one single experienced investigator blinded to group adherence. Comparison to age- and sex-matched randomly drawn controls adds further strength to the study. Some limitations must be mentioned. Since the participants are mostly Caucasians, the generalizability to other ethnic groups may be questioned. As the study had a population-based design and the population in Central Norway is almost exclusively Caucasians, we could not include more ethnic groups. The floor temperature of 5 °C resulted in a substantial number of censored cold pain threshold (CPT) responses. However, there were no group differences in the frequency of censored values, and we included statistical analyses accounting for censored responses. Due to the criteria for inactive disease and remission, internal subgroups of JIA only reflect disease status during the last year. Further, a challenge with all self-reported disease-related pain reports is whether the pain experienced by the participants are related and proportionated to their disease or might be influenced by other factors as, e.g., relationship with family and peers, level of education, physical activity, musculoskeletal co-conditions, and cultural or psychosocial factors. Different access to health care services may also influence on pain experience. In Norway, the health care system is mostly governmental and free of charge, so we do not think this will influence our results but may influence the generalizability to other countries with different health care systems than ours. Due to lack of information on socioeconomic status, the impact of this was not included in the analysis. Neurological diseases were not among the exclusion criteria, which could be a limitation. However, both participants with JIA and controls were asked if they had other diseases, but not specifically asked about neurological conditions.To our knowledge, this is the first study performing quantitative sensory testing (QST) in young adults with JIA. A few studies have been done in children and adolescents (6–17 years) with JIA with comparison to controls , 21–23. Another important issue regarding different responses to different test modalities is that pressure pain threshold (PPT) is the only quantitative sensory testing (QST) which reflects deep somatic pain corresponding to pain in JIA, in contrast to cold pain threshold (CPT) and heat pain threshold (HPT) which reflect superficial pain sensitivity , 41.In addition to QST studies, a Danish group investigating reaction to cold pressor pain has suggested reduced pain tolerance among children with JIA , 43. DueHeld together, all these studies suggest an altered pain sensitivity among JIA compared to controls. Our results mainly support this theory and furthermore expand the follow-up period into adult life.In adults with rheumatoid arthritis (RA), there are several studies including quantitative sensory testing (QST), and some with comparison to healthy controls , 44–49. Studies on associations between disease activity and quantitative sensory testing (QST) have shown inconsistent results. Hogeweg et al. found that individuals with active disease had lower pressure pain threshold (PPT) than those with inactive disease, but Leegaard et al. and Cornelissen et al. did not find this correlation , 21, 22.Increased suprathreshold pain response has been suggested as a sign of altered central sensitization . In our In accordance with our results, several studies show higher self-reported pain among individuals with JIA compared to controls , 50, 51.In conclusion, we found lower pressure pain threshold (PPT) and lower temperature equaling numeric rating scale (NRS) = 6 in young adults with JIA, compared to controls. Individuals with inactive JIA had the lowest PPT and cold pain threshold (CPT). No associations between self-reported pain and pain thresholds were found. This is the first study using quantitative sensory testing (QST) in young adults with JIA. Taken together, the study indicates for the first-time, altered pain perception and sensitization after long disease duration of JIA. A clinical implication of our study may be to emphasize the importance of early intensive treatment to quickly achieve pain-free remission and avoid long-term pain sensitization.Additional file 1: Supplementary Table S1. Thermal detection thresholds among juvenile idiopathic arthritis (JIA) and controls.Additional file 2: Supplementary Table S2. Median thermal and pressure pain thresholds in juvenile idiopathic arthritis (JIA) and controls.Additional file 3: Supplementary Table S3. Estimated differences in pain threshold per unit score in self-reported VAS pain.Additional file 4: Supplementary Table S4. Estimated differences in pain threshold: mild versus severe pain intensity / low versus high pain interference.


Dermestes dimidiatus ab. rosea was characterized using next-generation sequencing, and the phylogenetic relationships of superfamily Bostrichoidea were established. The results showed that the mitochondrial genome of D. dimidiatus ab. rosea was 16,073 bp in size, and it contained thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region. The composition of the whole mitochondrial genome of this species was 41.3% A, 13.5% C, 9.3% G, and 35.9% T, which had high A + T content (77.2%). Phylogenetic relationships of the superfamily Bostrichoidea showed that D. dimidiatus ab. rosea and Dermestes tessellatocollis formed in a clade that was a sister group to (Dermestes maculatus + Dermestes frischii), indicating that Dermestidae was a monophyletic group. This is the first report of a complete mitochondrial genome of D. dimidiatus ab. rosea and preliminary study of Bostrichoidea mitochondrial genome, which is of great significance for the molecular identification of this species and the enrichment of mitochondrial genome database.In this study, the complete mitochondrial genome of  Dermestes dimidiatus ab. rosea Kusnezova is a black and rose colored hairy beetle which belongs to superfamily Bostrichoidea. It distributes in former Soviet Union and Mongolia, as well as China including four provinces  and three Autonomous Regions   in Ningxia in June 2018. These samples were identified as D. dimidiatus ab. rosea, according to Zhang’s monographs (2016). Adults specimen of D. dimidiatus ab. rosea were preserved at −4 °C in 100% ethyl alcohol at Plant Quarantine and Invasion Biology Lab, China Agricultural University, Beijing. We used an adult beetle with the elytra removed and finished the DNA extraction using the DNeasy DNA Extraction kit (QIAGEN).The Berry Genomics sequencing company  finished the genomic DNA library preparation and sequencing. Genomic DNA was fragmented with Bioruptor to an average insert size of 450 bp and sequenced on an Illumina Hiseq 2500  was 16,073 bp in length, containing thirteen PCGs, twenty-two tRNA genes, two rRNA genes and a large non-coding region  for translation termination. Except for trnS1, all tRNA sequences could fold into the typical cloverleaf secondary structure. Particularly, the length of trnS1’s DHU arm was 0 bp, which was supported by Nie and Yang  and 1,273 bp (rrnL) in size. The control regionwas located between the trnI and rrnS genes with 1297 bp in length, while the overlapping sequences ranged from 1 to 35 bp in 18 regions, and the longest was between trnK and cox2.The Bayesian inference (BI) tree and IQ phylogenetic tree showed that Anobiidae and Bostrichidae consisted a sister group (D. dimidiatus ab. rosea and D. tessellatocollis formed in a clade that was a sister group to (Dermestes maculatus + Dermestes frischii).Adults nographs . Adults and Yang .Moreoverer group . The Derer group . For fam


Marchantia sp., Fissidens sp., Plagiochila sp., Sematophyllum demissum, Hypnum cupressiforme, and Calymperes motley, were subjected to sequential cold extraction with 3 different organic solvents. All three types of organic crude extracts were subjected to screening of antimicrobial bioassays using the disc-diffusion method against 3 bacterial strains and 1 fungal strain. According to the results obtained, 6 extracts out of 18 showed antibacterial activity for tested Gram-positive bacteria and 1 active against Gram-negative bacteria. Two extracts showed activity against the pathogenic fungus strain. Extracts from some plants were active against tested bacterial as well as fungal species. TLC-based bioautographic study was carried out to identify the corresponding active bands which is useful for active compound isolation. Furthermore, the ethyl acetate extracts were subjected to evaluate alpha-amylase inhibitory activity where three extracts out of six extracts showed moderate inhibitory activity for alpha-amylase with IC50 ranging 8–30%.Medicinal plants have been the main focus of natural product research. However, recent research has revealed that lower plants including bryophytes are also a major resource of biologically active compounds with novel structures. Sri Lanka is considered as a biodiversity hotspot with a higher degree of endemism flora including bryophytes. In this study, different species of bryophytes were investigated for their antimicrobial and alpha-amylase inhibitory activities. The air-dried plant materials of 6 different bryophyte species,  The contribution of natural products to the current pharmacopeia is significant while they have provided noticeable leads to novel drug discoveries . The exaSri Lanka is considered as a biodiversity hotspot comprised of a rich biological diversity of plant species with a high degree of endemism , 7. The Bryophytes are placed taxonomically between algae and pteridophytes and are divided into three classes: mosses, liverworts, and hornworts , 13. BryIn this study, 6 species of bryophytes from different ecological niches were collected and authenticated. The crude organic extracts, hexane, ethyl acetate, and methanol, were subjected to evaluation for their biological activities such as antimicrobial and alpha-amylase inhibitory activities.Whole plants of 6 plant varieties were collected from Kadugannawa (latitude of 7.255°N 80.5188°E), Kandy district, in the Central Province of Sri Lanka, on October 2019. The morphological and microscopic features of the fresh samples were recorded for authentication purposes.Due to the lack of herbarium specimens for lower plants, especially bryophytes, the plants were identified and authenticated by scientist Isuru Udayanga Kariyawasam, Molecular Plant Science (Phylogenomics of Bryophytes), at University of Edinburgh College of Science and Engineering, UK.Air-dried and pulverized plant materials were subjected to serial extraction with sonication using hexane, ethyl acetate, and methanol, respectively. The resulting extracts were filtered, and the filtrates were evaporated to dryness under reduced pressure using rotary evaporation  at 40°C. The resulted crude extracts were transferred into preweighted glass vials. The dry samples were stored at 4°C until further use.2HPO4), sodium dihydrogen phosphate (NaH2PO4), porcine pancreas alpha-amylase, NaOH, 3,5-dinitrosalicylic acid (DNS), sodium potassium tartrate, and distilled water.The chemicals and reagents used in this study were hexane, ethyl acetate (EtOAc), methanol (MeOH), 70% ethanol, sodium chloride solution, McFarland standard, 3--2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), disodium hydrogen phosphate (NaBacillus subtilis (UBC 344), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 9027) and human pathogenic fungi Candida albicans (ATCC 90028) were obtained from the Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura and Industrial Technology Institute (ITI), Sri Lanka.Human pathogenic bacteria Bacillus subtilis (UBC 344), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 9027) and one pathogenic fungi, Candida albicans (ATCC 90028), at 500 µg/disc concentrations using the standard agar disc-diffusion assay described by the American Clinical Laboratory Standard handbook with few modifications. In brief, cell suspensions of the test microorganisms equal to 0.5 McFarland were prepared using a 24 hrs old culture. A volume of 5 mL of each cell suspension was dispensed onto the surface of dried Mueller-Hinton agar , 38.0 g in 1,000 mL of distilled water) dishes, distributed all over the surface, and the excess suspension was removed (positive control: gentamicin (20 µg/disc); negative control: methanol). After overnight incubation, the mean diameters of the inhibition zones were recorded. Samples were dissolved in MeOH to make the final concentration 50 mg mL−1 accordingly. Then, 10 μL of the sample was delivered onto the sterile blank disc (WhatmanTM grade AA filter paper discs of 6 mm) to make the final concentration of 500 μg per disc. As the negative control, 10 μL of MeOH was delivered onto a sterile blank disc. Gentamicin (20 μg/disc) was used as the positive control for bacteria, while 1 : 1 mixture of ketoconazole and itraconazole (10 μg/disc from each) was used for pathogenic fungus. The growth inhibitions were visually examined by comparing with the positive control [The resulting crude bryophyte extracts were tested, in triplicate, for activity against three pathogenic bacteria,  control –19.μL of the enzyme and 40 μL plant extracts were mixed, and final volume of the reaction mixture was made up to 400 μL using phosphate buffer. Then, the mixture was preincubated for 10 minutes. After 10 minutes, 200 μL 1% of starch was added, and the mixture was incubated at 37°C for 30 minutes. After 30 minutes, the reaction was terminated by addition of 400 μL DNS to the mixture. Then, it was kept in a boiling water bath for 8 minutes and allowed to cool in a water bath. Absorbance was taken at 540 nm  after proper dilution with distilled water. Control experiments were conducted in an identical way, replacing the extract with 40 μL of DMSO as the negative control and 40 μL acarbose (10 mg/mL) as the positive control. For sample blank incubations , the enzyme solutions were replaced with buffer, and the same procedure was carried out.The EtOAc fraction of crude extracts was evaluated for their alpha-amylase inhibitory activities. The assay was performed according to the published protocol by Wickramarthne et al. with in-house modifications . pH 7.0,The results were expressed as % inhibition which was calculated using the following formula:S. aureus was used as the testing microbe since most of the extracts showed highest activity against S. aureus. As an exception, in this assay, the cell suspension was diluted to make the final OD (0.0001) by using melted MHA in 0.6%. The TLC plate was then overlaid with the diluted cell suspension of the test microorganism suspended in 0.6% MHA. Agar was allowed to solidify at room temperature and incubated at 37°C for 16–24 h. After the incubation period, in order to identify active bands, the TLC plate with MHA was flooded with a solution of MTT, 2 mg mL−1 (in sterile distilled water), and was incubated for 1 hr at 37°C.The extracts which showed promising antimicrobial activities were subjected to analysis using bioautographic techniques in order to identify respective antimicrobial active bands. Marchantia sp. (MR), Fissidens sp. (FS), Plagiochila sp. (PG), Sematophyllum demissum (SD), Hypnum cupressiforme (HC), and Calymperes motley (CM). The morphology of the above bryophytes is given in Less vulnerable bryophyte species were collected based on their availability. The collected bryophytes were identified based on macroscopic and microscopic morphological features. According to the features, the bryophyte species were authenticated as Fissidens sp. (FS-Hex) showed the activity against P. aeruginosa. Only hexane extracts of S. demissum (SD-Hex) and H. cupressiforme (HC-Hex) showed antifungal activity. None of the Plagiochila sp. extract showed any antimicrobial activity.In total, 18 extracts  were screened for their antimicrobial potential. The antimicrobial activities of crude extracts are given in S. aureus is given in The bioautographic analysis of the antimicrobial active extracts against Fissidens sp. showed the highest inhibitory activity (39%) followed by Marchantia sp. (23%). Two extracts (S. demissum and C. motley) were completely inactive.The alpha-amylase inhibitory activities of the EtOAc extract are given in Marchantia sp., Fissidens sp., Plagiochila sp., S. demissum, H. cupressiforme, and C. motley. There are two liverworts (Marchantia sp. and Plagiochila sp.), and the rest of bryophytes are mosses.Only a handful of studies have been carried out to investigate the bioactive potential of lower plants including bryophytes , 21, 22.Marchantia sp. are a good source of bioactive metabolites, and a number of compounds were isolated from different Marchantia sp. [Fissidens sp. and Plagiochila sp. are also good sources of secondary metabolites. Plagiochila barteri and Plagiochila terebrans are known to produce cyclic bisbibenzyls, ent-spathulenol, 1(10),14-halimadien-13ξ-ol, trifarienol B (11), and marchantins C and H, with antimicrobial, anticancer, and antiviral properties [S. demissum and C. motley. Therefore, this is the first record to investigate bioactive potential of these bryophytes.ntia sp. , 24. Marntia sp. –27. Fissoperties –30. HoweAccording to the disc-diffusion and bioautographic data of this study, most of the active compounds are in the nonpolar region. This is because bryophytes are lower plants, and they do not have special mechanism to preserve water. Hence, they produce different types of oil- and fat-based compounds in order to avoid loss of water, and most of the secondary metabolites of lower plants are lipophilic. The bioautographic analysis revealed the distribution of antimicrobial compounds. Normally, the bioautogram of higher plants is very complex and consists of several active compounds. However, the number of active compounds of the bryophytes used this study is limited to maximum 3. Therefore, isolation of antimicrobial compounds from these plants is quite easy compared to the higher plants. Furthermore, bioautography is a very valuable and time-saving method for bioassay-guided isolation of antimicrobial active compounds.Fissidens sp.; therefore, this is a valuable finding, and the extract can be used to isolate respective active metabolites since isolation of antialpha-amylase compounds from lower plants is very rare. Furthermore, alpha-amylase inhibitory activity represents in vitro antidiabetic activity. Hence, these active extracts are useful to regulate blood glucose levels and act as postprandial glucose regulators. There are only few studies on alpha-amylase inhibitory activity of bryophytes, and this study is one of them.Except 2 extracts, the other tested EtOAc extracts did not show potent inhibitory activity for alpha-amylase. The most promising inhibitory activity was shown by the EtOAc extract of Apart from the valuable finding of this study in the methodology section, all procedures are given in an explanative manner because most of the published articles mentioned these protocols briefly, and researchers are encountering problem while repeating these experiments. Therefore, researchers who are carrying out antimicrobial, bioautography, and alpha-amylase inhibitory assays can easily follow the given procedure.The results of this study show that the Sri Lankan bryophytes are capable of producing antimicrobial metabolites, active against both Gram-negative and Gram-positive bacteria, and antialpha-amylase activity and thus are potential sources for the discovery of new bioactive substances that may prove to be clinically useful. This is the first record of investigating bioactive potential of Sri Lankan bryophytes. Therefore, further studies on lower plants including bryophytes bring out their hidden biosynthetic capabilities, along with wider bioactivity testing which can be expected to immensely enhance the value of this valuable and underutilized resource.


Objective: Assess the variability and differences in oxidative stress, antioxidant, and inflammatory biomarkers in people with type 2 diabetes mellitus (T2D) and healthy controls.Methods: Ten men and women diagnosed with T2D and ten healthy matched controls (CON) were recruited. Participants had venous blood taken at six different time points on different days, three in the morning (after overnight fast) and three in the afternoon. Inflammation , oxidative stress/antioxidant biomarkers , glutathione peroxidase activity, IL-6, 8 & 10 and TNF-α) were assessed.Results: Biomarker concentrations were similar between groups. There was large variability in nearly all biomarkers for both groups. For inflammatory measures, intra-individual coefficients of variation (CV) ranged from 64.0–92.1% and 100.9–259.0% for inter-individual differences. CVs for oxidative stress markers were lower . TAC had the lowest intra-individual CV – 7% for T2D and 8% for CON. Protein carbonyls were more variable in the afternoon (34% CV) compared to morning (24% CV) in CON. IL-6 intra-individual CV was different between groups for afternoon measurements .Conclusion: Oxidative stress and inflammatory biomarkers show considerable variation in both T2D and healthy populations.Trial registration:ClinicalTrials.gov identifier: NCT01206725. Oxidative stress and inflammation share a cyclical link and are implicated in the pathogenesis of T2D ,2. Oxidain vivo; F2-isoprostanes, is contentious amongst scholars due to its reliability in determining lipid peroxidation without careful sample preparation and use of expensive complex instrumentation, such as gas chromatography mass spectrometry[Measurement of pro- and antioxidants has been a topic of debate amongst the literature for decades . Severalctrometry. Proteinctrometry.Knowledge of biomarker variability is crucial to determining perturbations from technical errors and biological noise . A great2-isoprostane isomer – 15(R)-prostaglandinF2α). We are unaware of any studies measuring oxidative stress biomarker variability in people with T2D. Therefore, the aims of this study were, in healthy individuals and people with T2D, to [Indeed, there is limited data on variability in oxidative stress and inflammatory biomarkers in healthy populations and people with T2D. One previous study conducted sampled urine daily over a one-month period in 19 healthy individuals . Intra-iThis is a sub-study of a randomised, controlled trial . Ten par2, pregnancy, drug, tobacco or alcohol abuse, planned surgery during the trial period, serious eating- and/or personality disorders, and reluctance to sign informed consent form. Participants were recruited through local newspaper advertising and from the outpatient population at St. Olav’s University Hospital, Trondheim, Norway.Inclusion criteria for the T2D participants were diagnosis of T2D less than ten years ago, non-insulin dependent, aged between 40 and 65 years, and achieving less than 210 min/week of exercise. Exclusion criteria included known cardiovascular or lung disease, uncontrolled hypertension, orthopaedic or neurological restrictions, body mass index (BMI) >35 kg/mUpon arriving at the laboratory in the afternoon, participants height and weight were measured, and a blood sample drawn from an antecubital vein. Participants were asked to avoid strenuous exercise for 24-hours prior to testing. Each participant was then asked to return to the laboratory for blood collection a further 5 times. Two afternoon collections and three morning collections on separate days over a two-week period.Ethylenediaminetetraacetic acid vacutainers  were used to collect all blood samples. Samples were immediately stored on ice before being centrifuged at 3000 rpm for 10 min at 4 degrees Celsius. Separated plasma was then aliquoted and stored at −80°C until biochemical assays were performed. Samples were shipped in dry ice to the University of Queensland, Brisbane, Australia for inflammatory and oxidative stress biomarker analysis.Plasma isoprostanes were analysed via gas chromatography mass spectrometry  as we have previously described in Briskey, Wilson . BrieflyPlasma protein carbonyls were analysed using an adaption of the Levine, Garland  method aPlasma glutathione peroxidase activity was measured using an adaptation of the method from Wheeler et al. Glutathione peroxidase activity was measured as the rate of oxidation of nicotinamide adenine dinucleotide phosphate, reduced (NADPH) at 340 nm in a coupled reaction, cycling oxidised glutathione to reduced glutathione using glutathione reductase. The reduced glutathione is utilised by glutathione peroxidase when the reaction is started with the addition of t-butyl hydroperoxide. These measures were performed on an auto analyser .Total antioxidant capacity (TAC) was measured using a modified version of an assay previously described, 20 and adapted for an auto analyser . Plasma was incubated with metmyoglobin and 2,2′-azino-biz (3-ethylbenzothiazoline-6-sulphonic acid). Post incubation hydrogen peroxide was added, and the sample was again incubated. Absorbance was measured spectrophotometrically to determine TAC.Plasma cytokines  were measured using the Milliplex MAP Human High Sensitivity T Cell Magnetic Bead Panel Kit. The assay protocol was followed as set out by Millipore, and magnetic beads were analysed by a multiplex analyser .Blood glucose, C-peptide, plasma triglycerides, total cholesterol, high density lipoprotein and low-density lipoprotein, high-sensitive C-reactive protein and HbA1c were analysed as previously reported in Hollekim-Strand, Malmo . InsulinLaboratory coefficients of variation (CV) for inflammatory and oxidative stress biomarkers have previously been developed by assaying 20 replicate samples. Laboratory CV’s are as follows; F2-isoprostanes – 7%, protein carbonyls – 11.9%, glutathione peroxidase activity – 2.4%, total antioxidant capacity – 1.9%, plasma cytokines – 10%.t-tests or Mann–Whitney U tests, where appropriate. The coefficient of variation (CV) is defined as the ratio of the standard deviation to the mean of a sample. CV was calculated as a measure of biomarker variability from the mean and determined the between- and within-individual (within each group) variability. Intraclass correlation coefficients (ICC) are used to describe the relationship between participants in each group and were calculated using log transformed within and between group standard deviations to determine the reliability of biomarkers [p < 0.05.All data analyses were completed using SPSS software . Normality testing of data using the Shapiro–Wilk test was performed. Between group differences were calculated using omarkers . Guideliomarkers . Sample omarker) . All datp < 0.05), however, groups were matched for all other characteristics.Participant characteristics for the patients with type 2 diabetes and control groups can be seen in p < 0.05). Diurnal differences for PC in CON were noted, with the CV in the morning 11% less than the afternoon sessions (p < 0.05). There were no between group differences in intra-individual biomarker CV when all timepoints were pooled together.Inter-individual variability is shown in The intraclass correlation coefficient (ICC) was calculated as a reliability measure for each biomarker . No biomThe aims of this study were to determine the day-to-day and diurnal variability, concentration and, consequently, reliability of inflammatory and oxidative stress biomarkers in people with type 2 diabetes and healthy controls. Our data show large between and within individual differences in almost all oxidative stress and inflammatory biomarkers for both healthy controls and people with T2D. These very high CV’s have considerable implications for their use in diagnosis, clinical decision making and research. Distinguishing a change in oxidative stress or inflammatory biomarker from ‘normal’ variation would be challenging at best.Interestingly, we found little difference in both the variability and the concentration of oxidative stress and inflammatory biomarkers between people with T2D and healthy controls. This is in contrast to much of the current literature suggesting that T2D pathogenesis is correlated with chronic low-grade inflammation and, by its cyclical relationship, also oxidative stress . We did 2-isoprostanes compared to this study cohort. Whilst the variability between individuals may be large this could be explained by biological individuality as previously discussed by Margaritelis, Theodorou [Previous research into oxidative stress biomarker variability by our group has reported similar within and between individual CV’s in F2-isoprostanes and protein carbonyls to this study . The varheodorou .ICC’s closer to 1 indicate that whilst there may be large between individual variability the within-individual variability is small, which is indicative of a more reliable biomarker. Contrary to expectations, this study did not find high ICC’s for any biomarkers measured . Indeed,d = 0.2) with our inflammatory and oxidative stress markers  assessing an increased sample size may provide further information towards determining whether reference ranges can be set for oxidative stress and inflammatory biomarkers.In summary, we report that people with T2D have similar variability of oxidative stress and inflammatory biomarkers to that of their matched controls. Interleukin-6 shows considerably more variability in people with type 2 diabetes, however this was found to be only in samples taken in the afternoon suggesting that there may be some diurnal variation in people with type 2 diabetes. Considering the variability observed in inflammatory and oxidative stress biomarkers in people with type 2 diabetes we recommend careful consideration of the sample size needed to detect both the minimal detectable difference between populations and clinically relevant results before commencing any further research. We caution the use of oxidative stress and inflammatory biomarkers in any clinical decision making for a type 2 diabetic population due to the high variability and poor reliability.


To evaluate the etiology, demographic profile, clinical features, and outcomes in patients with peripapillary subretinal hemorrhage (PSH).Thirty-eight eyes of 37 consecutive patients with PSH were enrolled in this prospective observational study over 4 years; all were followed for 2 years. The main outcome measures were demographic profile, possible etiology, clinical features, outcome, and prognosis.Sixty-eight percent (26/38) of eyes were in female patients; the mean patient age was 20 years. Only 1 patient (1/37) showed bilateral involvement. All patients experienced acute onset of PSH. All eyes were myopic and their best-corrected visual acuities ranged from 20/1000 to 20/12.5. The fundus features of affected eyes were classified into 3 groups: (1) PSH alone ; (2) PSH with intrapapillary hemorrhage ; (3) PSH with intrapapillary and vitreous hemorrhage . PSH occurred in nasal edges of optic discs with a crescent shape and dull-red color. All affected optic discs were small and crowded, exhibiting variable degrees of tilting. The cup of affected optic discs was narrower and deeper than that of normal control discs. Other ancillary tests provided no additional value. After a mean follow-up of 2.85 months, the hemorrhages resolved spontaneously without sequelae. Recurrence of disease was not observed in any patients.PSH is common in myopic eyes with tilted optic discs. We suspect that these hemorrhages occurred as a result of abrupt movement acting on a morphologically vulnerable optic disc. A clinical syndrome of peripapillary subretinal hemorrhage (PSH) with intrapapillary hemorrhage (IPH) has been reported by Cibis , Kokame Most cases have been reported in healthy young individuals of Asian ethnicities , 4, 6, 7Thirty-seven consecutive patients with isolated PSH, with or without IPH at presentation, were identified and evaluated from July 2012 through June 2016 in the Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, China. We excluded patients with optic disc edema, papillitis, ischemic optic neuropathy, optic nerve head drusen, juxtapapillary neovascular membrane, or glaucoma with optic disc hemorrhage. Patients taking corticosteroids or traditional Chinese herbs to relieve hemorrhage were also excluded.plus, Carl Zeiss Meditec AG, Jena, Germany), optical coherence tomography , and fundus fluorescence angiography . For differential diagnosis, other ancillary tests were performed in selected cases at the time of initial presentation; these tests included visual field test, B-scan ultrasonography, and auto-fluorescence of fundus (AF). B-scan ultrasonography (17 eyes) was performed to exclude drusen and to assess vitreous attachment to the disc. Fourteen eyes underwent visual field testing to exclude optic disc neuropathy. Six eyes underwent AF tests to differentiate retinal appearance from that of optic nerve head drusen. Three patients underwent computed tomography (CT) and 1 patient underwent magnetic resonance imaging (MRI) for head pathology.Medical and ocular history, ocular examination, and ancillary tests were meticulously performed for each patient. All patients underwent best-corrected visual acuity (BCVA) assessment, slit-lamp examination, dilated fundus evaluation with indirect ophthalmoscopy or with 90-diopter lens on slit-lamp, fundus photography , and 4 patients  had felt anxiety due to final examinations in high school. The male patient who had performed broad jumping exhibited bilateral involvement. We interpreted these events as risk factors . Statistical analysis of BCVAs between baseline and final visit for each group had been done separately. BCVAs in Group 2 and Group 3 showed significant improvement in vision (p < 0.05). Hemorrhages resolved spontaneously within 1 to 6 months (mean 2.95 ± 1.33 months). The mean durations of hemorrhage absorption in Groups 1, 2, and 3 were 2.00 ± 0.82 months, 2.53 ± 1.07 months, and 3.59 ± 1.42 months, respectively. In Group 3, which had vitreous hemorrhage, the duration of bleeding relief was longer than in Group 1 and no difference with Group 2  to 20/12.5 (LogMAR − 0.20)  and MRI (performed in 1/37 patients) outcomes were normal.When we analyzed the shapes of the optic disc and PSH, we found that they were obstructed by regions of hemorrhage in 25 eyes at the time of initial presentation. The optic discs of these patients were evaluated at the visit which hemorrhage absorption was complete. OCT images  and may even relax. This might explain why the bleeding is typically observed unilaterally.Our study has several limitations. First, the study designed without intervention is drawback. Second, although we performed study procedures on the first day of clinical visit, subjects in the study required extra time in or an extra trip to the clinic, not the initial time of onset, which may present bias on the clinical manifestation of inclusion subjects. Another potential limitation is that there were no control subjects. Considering the fact that anatomical peculiarity of optic discs is the basis, quantification from OCT and comparison with that of normal discs warranted further study.PSH syndrome is more common in younger patients with myopic eyes that exhibit tilted optic discs. It presents with variable clinical manifestations, such as PSH alone, PSH with IPH, and PSH with intrapapillary bleeding accompanying vitreous hemorrhage. Notably, PSH is not a single entity. We postulate that its causes may include sudden movement of myopic eyes with elevated optic discs. It is easy to misdiagnose PSH as optic disc edema, papillitis, buried optic nerve drusen, or other phenomena. The challenge for clinicians is to recognize the characteristics of PSH and distinguish this condition from others with similar features.


High levels of SGOT, SGPT and decreased platelet counts were found in severe patients as compared to Healthy donor. CRP/SAP as well as TNF-α/IL-1β were independently associated with both dengue severity and overall disease manifestation. Statistically significant increased CRP, SAP, ferritin, TNF-α and IL-1β titres were correlated in patients with severe clinical manifestations as compared to mild disease forms of dengue. Elevated levels of pentraxin, TNF-α/IL-1β in blood during dengue infection could act as an early predictor in Severe Dengue infection.Dengue is an arboviral infection with high rates of morbidity and mortality throughout the tropics and sub-tropics. This work studied the status of pentraxin (CRP/SAP) protein, ferritin, TNF-α and IL-1β levels in Dengue patients of different pathophysiological manifestations. Accordingly, clinically confirmed Dengue cases (n = 97) were enrolled and subsequently blood parameters were studied by Haematology cell counter and Biochemistry Autoanalyser. CRP, SAP, ferritin, TNF-α and IL-1β ELISA were done in all the samples by using standard ELISA kits. Statistical Analysis was done in all the experiments. The levels of CRP ( Severe dengue is the most dangerous form of dengue infection. Severe Dengue is characterized by a severe plasma leak resulting in shock, fluid accumulation with respiratory distress, severe bleeding assessed by the physician, severe damage to the organs of the liver, kidney, heart and other organs. Some patients have dengue fever without warning signs (DwoWS) and approximately 20 to 30% of them have dengue fever with warning signs (DwWS). During the last outbreak in West Bengal, a significant number of people were affected by significant morbidity and mortality. In addition to the usual hemorrhagic manifestations, significant morbidity has been observed in terms of organ involvement . Large number of patients have experienced gastrointestinal manifestations. More recently, Mandal et al., from the Calcutta Medical College, observed atypical characteristics such as transaminitis and various neurological manifestations present in 83.83% and 11.11% cases, respectively. So far, there is no adequate treatment, an antiviral drug, although some prospective solutions are currently being studied among different groups of scientists2. Early management, vector control and socio-educational programs are the only methods to reduce the burden of morbidity and mortality worldwide3.Dengue infection has become a serious problem in many countries, with a dramatic increase worldwide. It is estimated that more than 20,000 people die for dengue each year7.Currently, several tests can confirm dengue infection at the point of service. However, none of these tests can significantly predict the severity of the symptoms of the disease. During the onset of fever, it is difficult to distinguish between severe dengue fever and non-severe dengue fever. Since early hospitalization and supportive care can minify the case mortality rate of severe dengue fever, it is convenient to quickly identify patients who may develop severe dengue fever. Therefore, it is important to understand the immune pathogenesis of dengue infection to detect severe dengue fever at the beginning of infection. The pathogenesis of dengue infection is related to many factors, including the vector, the host genetic factor and the immune response9. In this study, we also investigate the status of another important vasodilating factor TNF-α. Interestingly IL-1β also involves the up-regulation of the pleiotropic cytokine IL-6, which is a key element in priming hepatic cells for APP production11. Further, TNF-α and IL-1β collectively/interdependently orchestrate the liberation of different chemokines and boosting the rapid and prompt recruitment of various leukocytes towards the inflammatory area. Hofmann et al., also demonstrated that TNF-α is also responsible for inflammation by inducing local vasodilation and vascular leakage in various infections.Colafrancesco et al. demonstrated that various acute-phase proteins play a crucial role to assess the asymptomatic inflammation. Acute-phase proteins (APP) are produced mainly by hepatic cells in response to various antigen-presenting cell-mediated cytokines, mostly IL-1β and IL-6. Previously our study also demonstrated that a high level of IL6 and vasodilation agent vascular endothelial growth factor (VEGF) were noted in severe dengue patients12. Previously, CRP had been reported as a beneficial biomarker in colorectal cancer13, rheumatoid arthritis15, coronary heart disease (CH)16 and chronic kidney disease (CRM) at the beginning of bacterial infection17, viral infection18, HIV infection19, parasitic infection of malaria20. In addition, Mairuhu et al., 2005 demonstrated the overexpression of pentraxin 3 in patients with severe dengue, but pentraxin 2 mainly SAP was not studied21. Subsequently, Ray et al. 201222 reported high SAP expression in patients with DF; However, its differential expression in patients with DWWS/SD (if any) remains unexplored. In addition, a strong expression of CRP in different degrees of dengue has also been reported21.Short pentraxin protein, such as C-reactive protein (CRP), Serum amyloid P are the oldest proteins that are positively regulated in a disease state in a very short time. CRP and SAP are synthesized by the liver during benign infections. An almost 1000-fold increase in CRP levels was recorded within four hours after the onset of the inflammatory disease like bacterial infection23 and Alzheimer's disease24. SAP proteins play a crucial role in bacterial infections25, viral infections26 and atherosclerosis27. As IgM antibodies against dengue may not be detectable until the fifth to eighth day of the disease and NS1 disappears from the blood much earlier in a secondary infection due to the presence of neutralizing antibodies, diagnosis becomes challenging. Taken together, in search of a biological marker of severity, we studied for the first time the status of inflammatory biomarkers such as CRP and SAP in patients with primary and secondary dengue infection.The serum amyloid P component, a pentameric glycoprotein, is also known as a diagnostic bio-marker for amyloidosisIn this study, 2772 patients suspected of having dengue fever were hospitalized at the OPD or were hospitalized at the beginning of infection at Carmicheal Hospital of Tropical Diseases, Kolkata, West Bengal, in India. Out of 2772 patients, 97 (3.49%) were positive for NS1/IgM/IgG anti-Dengue antibody. Twenty healthy subjects and 20 other febrile infection like malaria, chikungunya served as study control and were designated HD and OFI respectively. On the basis of clinical manifestations, 26 were classified as DWWS and 9 patients with SD. Classification was based on clinical history and clinical characteristics of selected patients, followed by WHO guidelines. Among the patients infected with dengue, approximately 43.29% (n = 42) were men and 56.69% (n = 55) were women. Out of the 97 patients with confirmed dengue fever, 41 patients were classified as secondary dengue , DwWS (n = 11), and SD (n = 3))  had fever, myalgia , rash , headache ), vomiting  and abdominal pain  Table .p < 0.0001), platelets (p < 0.0001), globulin (p = 0.148) and hematocrit (p < 0.0001) showed a significant difference , SAP(p < 0.0001), ferritin (p = 0.0004), biochemical parameter SGOT (p = 0.024), SGPT (p = 0.029) and platelets (p = 0.007)  of SGOT  and SGPT  were detected in the sera of patients with DwoWS. In patients with warning signs of the disease, SGOT levels  and SGPT  were lower than those of patients with SD . SD patients had decreased thrombocyte counts approximately fourfold and 2.18-fold, respectively as compared to DwoWS and DWWS. In addition, ANOVA analysis between DwoWS, DWWS and SD with respect to SGPT  Figure .Figure 1p < 0.0001) and DWWS (p < 0.001)   and DWWS (p < 0.001) .Initially, circulating levels of CRP were evaluated in different patients with dengue, i.e. DwoWS , DWWS  and SD  ml) compared to HD  were significantly higher in patients with severe dengue. However, CRP levels in OFI were very low  as compared to SD . Now in this study we also observed high levels of TNF-α in SD , DWWS  and DWoWS  as compared to HD . Hence, 3.37 fold increased TNF-α was observed in SD patients as compared to DWoWS. However,TNF-α levels in OFI were very low as compared to SD (p < 0.0001).The levels of circulating cytokines were also analyzed in context to primary versus secondary infections Figs. C and 6C.p = 0.001), SAP (r = 0.273 with p = 0.01), while the platelet was negatively correlated with CRP (r =  − 0.598 with p < 0.0001). Interestingly, the platelet was found to negatively correlate with SAP levels (r =  − 0.0284 with p = 0.008). These data indicate a possible influence of these acute phase proteins on elevation of SGOT and painful liver enlargement. Further, serum cytokine levels TNF-α was positively (r = 0.376 with p = 0.0005) and negatively (r =  − 0.556 with p < 0.0001) correlated with SGPT and platelet respectively. On other hand, elevated liver SGPT levels was positively correlated with IL-1β (r = 0.363 with p = 0.0009), while the platelet was inversely correlated with IL-1β (r =  − 0.390 with p = 0.0003). These association indicate that IL-1β and TNF-α probably induces liver tissue injury, vascular damage and thrombocytopenia . Out of the 9 patients with SD infection, 77% suffered from whole abdominal pain and liver problem in this study. These patients had higher levels of CRP and SAP compared to those for whom symptoms were absent. The increase in SGOT levels was positively correlated with CRP . CRP belongs to the family of pentraxin proteins and is used as a biomarker of inflammation, especially for bacterial infections29. In general, it is assumed that viral infection increases CRP levels by 10 to 40 mg/L31. In our study, the median CRP level was significantly elevated in people with severe dengue compared with the less severe form (DwoWS/DWWS). The critical phase of dengue occurs more frequently towards the end of the febrile stage, usually after the third day of the disease32. In particular, we found a significant association of higher levels of CRP with severe dengue fever during the first 3 days of the disease (febrile phase). Previously, Mairuhu et al., 2005, had shown that the level of CRP was very high among people with severe dengue compared to non-severe dengue in the population of the Netherlands21. However, the status of CRP in severe secondary dengue remains unexplored in the population of eastern India. Pentraxin protein SAP have also not been studied. Subsequently, Ray et al. 201222 reported high SAP expression in patients with SD; however, its differential expression in patients with DWWS/SD (if present) remains unexplored. In addition, high levels of SAP have been reported in dengue infection, which play an important role in the opsonized pathogen or in other cells that are dead due to their phagocytic clearance33. It is interesting to note that in this study, significantly elevated levels of pentraxin proteins were observed in SD compared to DWWS . As the vascular permeability, bleeding, ascitis are the main symptoms of SD patients, we evaluated the levels of IL-1β in dengue patients at the early stage of infection. Here we found very high levels of serum IL-1β in SD patients as compared to DWWS (p < 0.001) however the levels of IL-1β in DWoWS were negative as compared to HD. This data suggested that high levels of IL-1β in SD patients could play crucial role in its pathogenesis. Previously JR Bethea et al., 1992 demonstrated that IL-1β induced the production of TNF-α, from the monocyte34. TNF-α is mufti-functional cytokine that could affect on endothelial cells35 and take part in certain manifestations of DWWS and SD, such as low platelet count, internal or external hemorrhage, ascitis etc.36 Although DWoWS patients were negative for TNF-α, but elevated levels have been observed in DWWS and SD compared to that of healthy donor (p < 0.0001). These data support that TNF-α production in SD and DWWS patients is connected to immuno-pathology of severe dengue infection. To our knowledge, the comparative study of serum pentraxin (SAP/CRP) protein, ferritin, TNF-α and IL-1β levels in patients with severe secondary and primary dengue fever in eastern India has not yet been well studied and our current study will help to initiate more studies to unravel the potential of pentraxin protein as a biomarker in dengue severity.Dengue fever is an unpredictable viral disease. Early identification and management of severe dengue patients are very crucial step to prevent death rate until the discovery of anti dengue drug. To our knowledge, no specific study has been conducted to date on the pentraxin profile in primary and secondary dengue fever in Kolkata, a hyperendemic dengue zone in eastern India. In this study, we studied the status of inflammatory proteins in the acute phase in patients with dengue. Many host as well as pathogen related factors contribute to the pathogenesis of the dengue virus. Several components are produced from hepatic cells infected with the dengue virus, such as CRP, SAP and ferritin in the early stage of infection. The first described acute phase protein (APP) was C-reactive protein (CRP), a positive APP identified in humansTaken together, all these acute phase proteins and TNF-α/IL-1β increase the severity of the disease. Given the important role played by APP and cytokines in the pathogenesis induced by viruses, its association with different biochemical parameters was also studied. All clinical manifestations, joint pain, retro-orbital pain, headache and muscle pain were common to all patients with dengue fever. On the other hand, it has been shown that vomiting, abdominal pain and organ damage are positively associated with SD and could be used as prognostic symptoms of SD. Organ involvement, specially liver has been well documented in severe form of dengue infection. In addition, in our study, biochemical parameters such as high liver enzyme like SGPT, SGOT, albumin, globulin, haematocrit and low thrombocyte count were well documented in patients with SD as compared with DwoWS. However, a slight statistically significant difference was ascertained in the routine haematological as well as biochemical test between patient with severe dengue and HD only for SGPT, SGOT, platelets and APP/cytokines on day 3 of the infection. Therefore, according to biochemical parameters, it was difficult to distinguish SD from DwoWS in the early stages of infection. Therefore, we quantified and established a specific threshold value for the APP and cytokines that could provide us with a clear indication of the severity of dengue fever. Therefore, our research revealed that among the APP analyzed, based on the specific cut-off value of CRP, ferritin, TNF-α and IL-1β it could act as a severity biomarker. Inflammatory APP, such as increased CRP/SAP, has also been significantly associated with decreased platelet counts and increased SGPT in patients with severe dengue. Hepatomegaly in patients with severe dengue was well documented, which was reflected again in an increase in SGOT and serum APP. Similarly TNF-α and IL-1β was also associated with platelet destruction and hepatic cell injury. Our data revealed that most severely ill patients have accumulation of fluid in the liver and lungs; Decrease in the number of platelets, increase in APP titres. The two APPs were found to have slightly higher titers in secondary infections than in primary infections. The data from this study reveal that the increase in pentraxin proteins are well related to the severity of dengue fever in the first days of infection. The study limitation is the evaluation of these data in a much larger patient population should be used before a global clinical application.The Clinical Research Ethics Committee, Calcutta School of Tropical Medicine, West Bengal, Kolkata, India, on Human Ethics approved the study  and took the necessary blood samples from the healthy control and patients. A written informed consent was obtained from both enrolled patients and the healthy control population. All methods were performed in accordance with the relevant guidelines and regulations.This was a hospital based control study. Dengue virus infected patients diagnosed at Calcutta School of Tropical Medicine (CSTM), West Bengal, India during August 2017 to September 2018 were enrolled. Patients with dengue-like disease (n = 2772) were examined and four milliliters (4.0 ml) of venous blood were collected by venipuncture and retained in EDTA anticoagulant tubes. Plasma was separated by centrifugation at 3000 rpm and subjected to NS1 MAC-ELISA, IgM/IgG ELISA . Out of the 2772 patients suspected, 97 NS1 and IgM/IgG were selected for our study, of which 62 were classified as dengue without warning signal (DwoWS) and 26 patients with the same type of dengue with warning signal (DWWS) and 9 others were severe (SD). In addition, 20 healthy donor (HD) and 20 other febrile infection (OFI) like malaria, chikungunya subjects were also enrolled for this study. Serum was collected for all subjects in the study and stored at − 20 °C until further use.To characterize the pathological state of the study subjects, a detailed biochemical analysis of the blood samples was measured using a standard auto-analyzer (ERBA model No. EM360). These include serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), globulin and albumin. In addition, white blood cell counts, red blood cell counts, hemoglobin (HB), hematocrit (HCT), and platelet count (PLT) were measured using an automatic cell counter. (model SYSMEX No -KX100). The tests were performed following the manufacturer's provided instructions. All samples were analyzed and the value was taken for analysis.To quantify acute serum protein levels in different groups of dengue infections, the stored sera were thawed and CRP, SAP and ferritin were evaluated using standard ELISA kits . The ELISA tests were performed according to the manufacturer's instructions (Ray Biotech). The samples were analyzed and the median value was taken for analysis.To quantify inflammatory and anti-inflammatory serum cytokines levels in different groups of dengue infections, the stored sera were thawed and TNF-α and IL-1β were evaluated using standard ELISA kits . The ELISA tests were performed according to the manufacturer's instructions (Ray Biotech). The samples were analyzed and the median value was taken for analysis.9. Briefly, all data were subjected to ANOVA with post-hoc test for finding the significance of each study groups. Further, Mann–Whitney test was performed to estimate the statistical significance between each group. The correlation between APP/Cytokines and blood parameters was determined by using Spearman's correlation.Statistical analysis was performed using the Graph-Pad Prism statistics software  and all statistical methods were adopted from Patra et al., 2019Supplementary Information.


Turbulent blood flow in patients with aortic valve stenosis (AS) results in morphological and functional changes in platelets and coagulation factors. The aim of this study is to determine how shear stress affects platelets and coagulation factors.We retrospectively evaluated data from 78 patients who underwent AVR to treat AS between March 2008 and July 2017 at Kagoshima University Hospital.Platelet (PLT) count obviously decreased at three days after AVR, and increased above preoperative levels at the time of discharge. In contrast, platelet distribution width (PDW), mean platelet volume (MPV), and platelet large cell ratio (P-LCR) increased three days after AVR, then decreased to below preoperative levels. No differences were evident between groups with higher (HPPG > 100 mmHg) and lower (LPPG < 100 mmHg) peak pressure gradients (PPG) before AVR, whereas PLT count, PDW, MPV and P-LCR improved more in the HPPG group. Plateletcrit (PCT), which represents the total volume of platelets, increased after AVR due to decreased shear stress. High increasing rate of PCT was associated with lower PLT count, higher PDW and lower fibrinogen.Shear stress affects PLT count, PDW, and fibrinogen in patients with AS. AVRAortic valve replacementASAortic valve stenosisPPGPeak pressure gradient(s)HPPGHigh peak pressure gradient(s)LPPGLow peak pressure gradient(s)MPVMean platelet volumePCTPlateletcritPLTPlatelet(s)PDWPlatelet distribution widthP-LCRPlatelet large cell ratioTAVITrans-catheter aortic valve implantation1The prevalence of cardiovascular diseases associated with age-related vascular atheroma, intimal thickening, and calcification is increasing globally. Among valvular diseases, the number of patients with aortic valve stenosis (AS) caused by calcification has also increased, and it is expected to double within the next 20 years. AS is generally treated by replacing a stenotic aortic valve with a prosthetic valve, which requires a cardiopulmonary bypass during the procedure and causes problems for older patients. Trans-catheter aortic valve implantation (TAVI) is less damaging for patients with AS. However, this is sometimes contraindicated due to anatomical restrictions and financial issues. At present, the pathological mechanisms of AS and valve calcification have not been sufficiently elucidated, and preventive agents are unknown. Valve calcification gradually progresses in AS, and the area of valve orifices decreases over time. As the pathological lesion advances and the valve orifice area decreases, blood flow passing through the aortic valve becomes turbulent , and peain vivo and in vitro [β) by activating platelets in mice AS model [Shear stress due to blood flow acts in a tangential direction to the surface of blood vessels and increases according to blood viscosity and flow velocity . Shear sin vitro . For insAS model . PlateleAS model , howeverAortic valve replacement (AVR) normalizes turbulent blood flow caused by AS, and should thus alter force and direction of shear stress. That is, shear stress before and after AVR might be quite different, and exert various effects on blood cells. This study analyzed changes in the peripheral blood before and after AVR and examined the influence of shear stress on blood cells, especially on platelets.22.1We retrospectively investigated 78 patients who underwent AVR only at our hospital between March 2008 and July 2017. We collected blood test results, clinical information about the patients, and transthoracic echocardiography data. Peripheral blood data were collected before surgery (T1) and on postoperative days (POD) 3 (T2) and 7 (T3), and on the day of discharge (T4). Biochemical and coagulation data were collected at T1 and T4. The first postoperative month is described as T4 for patients who remained in hospital for more than one month. We compared white blood cell (WBC) count, red blood cell (RBC) count, RBC distribution width (RDW), platelet (PLT) count, platelet distribution width (PDW), mean platelet volume (MPV) and platelet large cell ratio (P-LCR) in peripheral blood samples. The patients were assessed by transthoracic echocardiography before surgery. We measured the PPG, mean pressure gradient (MPG), aortic valve area and left ventricular ejection fraction (LVEF). No case was excluded to use analyzed data in this study. This investigation proceeded under approval from the Kagoshima University Hospital Clinical Research Ethics Committee . The study was conducted in accordance with the ethical standards of the Committee on Human Experimentation of the institution at which the experiments were performed or in accordance with the ethical standards of the Helsinki Declaration of 1975.2.2χ2 tests, Student t tests or Mann-Whitney U tests, as appropriate. Values with p < 0.05 were considered to be statistically significant.Data were statistically analyzed using GraphPad Prism 8 . Values were expressed as means±SD. Groups were compared using 3We assessed data from 78 patients with AS, including 13 who were on hemodialysis. μL at T1 to 10,772±3,847/μL on T2 and gradually decreased to 6,534±2,242/μL at T4, but not to the preoperative level , increased to 18.0±6.65×104/μL at T3, and reached 25.5±8.21×104/μL at T4, which exceeded that at T1  = 4× (velocity (m/s))2) and we assigned the patients into groups with HPPG  and LPPG . Four platelet factors  were not changed at T1 in the two groups  can be calculated from flow velocity according to the simple Bernoulli equation (o groups . Althougo groups . In conto groups . In PG c4/μL)×MPV (fL)×10–3), which represents the total platelet volume in the blood, before and after surgery. The transition of PCT had no significant difference between HPPG and LPPG (Supplementary 3/μL and 20.0±4.58×103/μL (p < 0.001) in the groups with high and low PCT increasing rate, respectively. In contrast, PDW was 12.6±1.74% and 11.8±1.41% (p = 0.04) in the groups with high and low PCT increasing rate, respectively. Neither MPV nor P-LCR significantly changed . These data suggest that high PCT, which should indicate high shear stress, results in smaller PLT count and larger PDW before AVR. In other words, the influence of shear stress to platelets was more powerful in patients with low PLT count and a large PDW before surgery.To establish a new factor with which to predict the extent of shear stress, we calculated plateletcrit (PCT (%); PLT count (10p = 0.06), different changes between the groups , and activated partial thromboplastin time (APTT). Whereas PT and APTT did not significantly differ between patients with high and low PCT, the fibrinogen value was lower in the group with high PCT A. The AS4We assessed the dynamics of blood cells in patients with AS treated by AVR during the perioperative period. PLT count recovered after surgery, and PDW, MPV, and P-LCR, which represent variations in platelets, decreased compared with those before surgery. The preoperative PLT count was low and PDW was wide in the group with high PCT from which we predicted high shear stress. Among the patients with AS, PCT was a little lower for those on dialysis after AVR. Furthermore, fibrinogen was significantly low in the group with high PCT, suggesting a relationship between shear stress and coagulation ability in patients with AS .The frequency of bleeding increases after cardiac surgery due to the complexity and length of surgery and the need for a cardiopulmonary bypass. Prolonged postoperative bleeding requires more surgery, which negatively impacts mortality and increases the likelihood of further complications . VuylsteThe intima of the blood vessels is constantly exposed to hemodynamic forces, namely vertical pressure from the circulating blood and fluid shear stress caused by tangential force and blood viscosity on the surface. Shear stress within the physiological range is required to maintain vascular function, but excessive shear stress damages blood vessels and blood cells , and is Shear stress activates leukocytes, leading to chronic inflammation in patients with AS . We showOn the other hand, the PLT count was higher at the time of discharge than before the operation, and other factors were lower than the preoperative value, and the change in platelets was larger than that of other blood cells. Regarding the blood of patients with AS, it was reported that the platelet and vWF activity was reduced before the operation and improved after the operation . It was Fibrinogen was low in high rate of increase PCT group, indicating that shear stress might influence coagulation ability. Natorska et al. have found high values for thrombin and PLT activities in some patients with AS , and sug2 activates circulating platelets. Since the estimated shear stress is 1,000–1,700 dyn/cm2 in AS mimic model [β that promoted cell transformation in aortic valves [Shear stress between 100 and 1,000 dyn/cmc valves  and enhac valves . Shear sc valves . In turnc valves . Taken tAVR for AS requires a prosthetic valve, and mechanical or bioprosthetic valves are presently available, and the mechanical type requires the lifelong medication with warfarin, which causes vascular calcification . Althoug5This retrospective study is limited by the protocol design and a small patient cohort. Although restricted to patients who underwent AVR only, preoperative factors that could affect blood cells was impossible to standardize from their backgrounds. Moreover, since the patients were not followed up at our institution, changes beyond one month of follow-up could not be compared. We consider that high postoperative WBC counts and CRP values are due to a persistent inflammatory response to invasive surgery. Since inflammation might have affected blood cell counts and morphology, accurate longer-term comparisons of the effects of shear stress are warranted.6The present findings indicated that shear stress activated PLT, which were consumed by forming micro-thrombus and PCT might be a marker of shear stress in AS patients. The relationship between PCT and platelet function should be investigated in the future study.The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the following; Challenging Research (Exploratory) (18K19523) (M.Y.), Grant-in-Aid for Scientific Research (18H02734) (M.Y.), (16H05229) (T.H.), (19K09274) (Y.I.), (17K09310) (Y.O.), Grant-in Aid for Early-career scientists (18K16399) (H.K.), and (18K15424) (K.T.).


This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium.Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%.Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp. count.According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable The online version contains supplementary material available at 10.1186/s12866-021-02109-1. Legionella pneumophila, which represents the etiological agent responsible for Legionnaires’ disease (LD), was first isolated by McDade et al. in [Legionella infection have been reported to be associated with water distribution systems, air conditioning devices, spas and cooling towers [Legionella spp. is now being recommended worldwide.t al. in . After ig towers , 21, 22.Legionella species comprising 70 distinct serogroups have been identified to date (https:/l-1psn.dsmz.de/genusl-1egionella). Most Legionella species have been isolated from aqueous environments, with at least 30 of them capable of causing infection in humans, mainly in the lower respiratory tract [More than 66 Legionella is a particularly fastidious gram-negative bacterium: in the late 1970s, Feeley and Gorman prepared a new agar medium, cysteine F-G agar-iron, with L-cysteine hydrochloride and soluble ferric pyrophosphate [Legionella spp. Later on, Pasculle et al. supplemented the CYE medium with N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer, thereby obtaining buffered charcoal yeast extract agar (BCYE) which, under aerobic conditions, enabled a better recovery of Legionella [hosphate , while Fhosphate  and the gionella , and Edegionella .Wadowsky–Yee (MWY) medium [Legionella spp. via reduction of non-Legionella bacteria, most protocols, i.e. Health Protection Agency [Legionella spp. due to overgrowth or inhibition [Since then, numerous selective media based on different inhibiting agents have been developed so as to limit the development of interfering microbiota that may reduce or inhibit the recovery of these bacteria. Notable among these are GVPC agar, a BCYEα medium supplemented with 3 g/l glycine, 0.001 g/l vancomycin, 80,000 IU/l polymyxin B and 0.08 g/l cycloheximide, and the modified ) medium , 32, anon Agency , AFNOR [n Agency  and Centhibition .Legionella spp. testing on environmental samples using two types of medium: BCYEα and MWY [Legionella detection has been recently (2017) recommended by the second edition of ISO 11731 [Legionella isolation: GVPC or MWY agar for water samples with a high concentration of interfering microbiota, and BCYEα +AB agar  for samples containing low concentrations of interfering microbiota. This latter selective medium was first recommended by the Dutch Standard NEN 6265 [Since 1997, our laboratory has been conducting  and MWY . We adop and MWY . The comcently 20 recommenThe goal of this study was to compare the performance of the BCYEα +AB vs. MWY agar in terms of sensitivity and selectivity in suppressing the growth of interfering microbiota from water samples taken from hospital water supplies.Legionella spp. Of these, 143 (54.4%) were Legionella positive at least on one medium. The distribution of the results between the two media is shown in Table A total of 263 hot water samples, all taken from hospital potable water faucets, were cultured on two different media  to isolate Legionella in our samples (and enumeration of Legionella spp. colonies for positive samples) was possible for 98.1% (258/263) of the samples seeded on MWY agar and 95.4% (251/263) of those seeded on BCYEα +AB agar, whereas all samples contaminated by overgrowth of Gram-negative bacteria were deemed as Legionella negative.A small minority of the specimens (5/263) contained Gram-negative bacteria that were not inhibited by either selective medium , while 7 further samples contained flora whose growth was inhibited by the MWY selective medium but not by BCYEα+AB Table . TherefoAgreement between the two methods was 86.7%. Calculation of Cohen’s κ-coefficient showed good concordance (κ = 0.733) Table .Table 2Table L. pneumophila (59.4%) and was detected equally using the two agar media. In 29 of the 143 positive water samples, L. pneumophila grew with associated non-pneumophila Legionella species. The most frequently isolated serogroups were L. pneumophila serogroup 6 and serogroup 1. The distribution of the results is shown in Table The most frequently isolated species was phila 59.% and wasLegionella spp. positive on MWY agar, 23 showed the presence of accompanying microbiota (18.5%), whereas accompanying microbiota was observed in 52 out of 127 Legionella spp. positive samples (40.9%) on BCYEα+AB agar . The same analysis yielded a statistically significant difference in non-Legionella flora growth between the MWY and BCYEα +AB media (p < 0.0001). Furthermore, the counts from the 108 positive samples on both agars were compared by evaluating Kendall’s tau correlation coefficient, which showed a high degree of correlation   [Legionella spp. [.la spp.) , 19. In lla spp. .Legionella species: GVPC or MWY agar for water samples with a high concentration of background flora and BCYEα+AB agar for samples with a low concentration of interfering microorganisms. However, given that the selective culture of Legionella may also be influenced by the degree of susceptibility of the microorganisms to antimicrobials, which varies according to their physiological state, here we inquired whether the use of BCYEα +AB agar could improve the sensitivity of Legionella spp. detection in environmental water samples over that of current methods. To our knowledge, this is the first study addressing detection and enumeration of Legionella species on BCYEα +AB medium.More recently, the new edition of ISO 11731 [Legionella CFUs on BCYEα +AB agar was not significantly higher than the number of CFUs found on MWY agar  (p = 0.09934), indicating that there are no significant differences in sensitivities between the two selective procedures. Thus, our results demonstrate that both selective media are suitable for primary plating of environmental specimens for the isolation of Legionella spp.Agreement between the results obtained with the two media was 86.7%. Importantly, parallel seeding showed that the number of Legionella spp. cells that are harmed or not that much active in the water systems would not grow. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and / or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella.According to the results, both methods have a very similar performance and they both have advantages and disadvantages over each other. In AB media there is the risk of being less selective so more interfering microbiota may grow but in MWY media there is the risk of being too selective and Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp. count , and onto BCYEα+AB , consisting of natamycin (65 mg/l), cefazolin (9 mg/l) and polymyxin B .Analyses for the quantification of Legionella spp. were performed according to an internal method . Water s2 for 10 days. The plates were checked at days 2, 3, 5 and then at the end of the incubation period. Presumptive Legionella colonies were confirmed by subculturing on blood agar  and BCYEα agar. Colonies grown on MWY agar or BCYEα+AB agar were identified according to ISO 11731 procedure  by means of an agglutination test . The latex test allows separate identification of L. pneumophila serogroup 1 and serogroups 2 to 14 and detection of seven other non-pneumophila Legionella spp.  [Legionella spp., as previously described [Legionella spp. in the original sample. Legionella spp. concentrations in water samples are expressed in colony forming unit per liter (CFU/l). According to the concentration procedure, the detection limit of our method is 50 CFU/l. The presence of background flora was measured through semi-quantitative counting [Briefly , the one. anisa) , 25, 31.escribed . The placounting : four catau correlation coefficient was employed to compare the ability of the two media to cultivate Legionella. The analysis was performed by taking into account only samples positive for Legionella spp. on both media, after checking the preliminary assumptions.Statistical analyses were performed using the statistical software R  . AgreemeAdditional file 1. Background flora. Examples of plates with different level of background flora from complete absence (zero) to massive contamination (3+).Additional file 2. Paired photos of MWY BCYEα+AB obtained during incubation period of inoculated plates. Examples of Concordant Positive Samples (MWY agar on the left and BCYEα+AB agar on the right) . Samples Positive Only on MWY agar (pag 3). Samples Positive Only on BCYEα+AB agar (pag 4). Examples of Samples With Overgrowth only on Bcye+Ab Agar; Examples of Samples With Overgrowth on Both Agar Media (pag 5).


Kawasaki disease (KD) is a systemic vasculitis, and the formation of coronary artery lesions(CAL) is its most common sequela. Both genetic and environmental factors are considered to be important factors of in KD. Integrin α2 (ITGA2) is a transmembrane receptor that is associated with susceptibility to several diseases, but its relevance to KD with CAL is unclear.We genotyped ITGA2 rs1126643 in 785 KD patients with the CAL and no-CAL(NCAL) . OR (95% CI) and adjusted OR (95% CI) were used to evaluate the intensity of the association.P = 0.0032; CT/TT vs CC: adjusted OR = 1.49, 95% CI = 1.12–2.00, P = 0.0068; T vs C: adjusted OR = 1.66, 95% CI = 1.16–2.51, P = 0.0165). Moreover, we found that carriers of the CT/TT genotype had a significant risk of KD with coronary artery lesion susceptibility for children ≤60 months of age, and the CT/TT genotype was significantly associated with an increased risk of SCAL formation and MCAL formation when compared with the CC genotype.We found a significantly increased risk of KD with CAL associated with ITGA2 rs1126643 genotypes (CT vs CC: adjusted OR = 1.57, 95% CI = 1.16–2.12, ITGA2 rs1126643 was associated with increased susceptibility and severity of CAL in KD. Kawasaki disease (KD) is a systemic vasculitis first described by Dr. Kawasaki in 1974 in Japan , 3. The Antiplatelet therapy is routinely applied in KD treatment. Elevated platelet counts often develop in the acute phase of KD. This causes hyperplatelet function in which platelets become adherent, deformed, aggregates and release various cytokines and inflammatory factors. As a consequence, blood hypercoagulates causing vascular endothelial injury, collagen exposure, and triggers chemotaxis of various cytokines, all of which results in the formation of vascular inflammatory lesions . Low-dosA total of 300 patients with KD coronary artery lesions, diagnosed in line with the criteria of the American Heart Association in 2004  who wereGenomic DNA was extracted from peripheral blood samples for genotypic analysis of the SNPs rs 11,226,643) of interest in the ITGA2 gene. Functional polymorphism was carried out based on a thorough evaluation of polymorphisms associated with vasculopathy , 11. Thi,226,643 2 test. The association of the ITGA2 rs1126643 C > T polymorphism with KD susceptibility was evaluated by calculating the odds ratio (OR) and 95% confidence interval (CI) using unconditional multivariate logistic regression analyses. Stratified analysis was based on coronary artery outcomes, age and sex, and P-values < 0.05 were considered statistically significant. Statistical analyses were performed using SAS software .The frequency distribution of the polymorphism and the demographic variables between KD with CAL and NCAL were compared using the χP = 0.2411) or sex (P = 0.0603) between CAL and NCAL groups. Two-dimensional echocardiography revealed that 188 (62.67%) patients developed coronary artery dilatations/small aneurysms (SCAL), 63 (21.00%) developed coronary artery medium aneurysms (MCAL), and 49 (16.33%) developed giant coronary artery aneurysms (GCAL).The demographic characteristics of the KD patients with and without CAL are summarized in Table P = 0.0032; CT/TT vs CC: adjusted OR = 1.49, 95% CI = 1.12–2.00, P = 0.0068; T vs C: adjusted OR = 1.66, 95% CI = 1.16–2.51, P = 0.0165). No other significant associations were detected. As a result, the harmful genotype used for the calculation was ITGA2 rs1126643 CT/TT.The frequency distribution of the SNP polymorphism in the Kawasaki disease patients with and without CAL is shown in Table P = 0.0199) and males . The CT/TT genotype was significantly associated with an increased risk of SCAL  and MCAL  compared with carriers of the CC genotype.The association of ITGA2 gene rs1126643) polymorphism with KD coronary artery lesion susceptibility was further determined in stratified analysis by age, sex and severity of CAL  polymorphism with the risk of KD in 300 patients with CAL and 485 patients with NCAL. Specifically, the ITGA2 rs1126643 CT/TT was associated with an increased risk of KD patients with CAL, especially in children ≤60 months of age and males. Stratified analysis demonstrated that the ITGA2 rs1126643 CT/TT variant elevated the risk of KD progressing into SCAL and MCAL. To our knowledge, this is the first study to show that ITGA2 rs1126643 polymorphism is associated with KD coronary artery lesion susceptibility.Integrins are adhesion molecules that promote platelet aggregation, hence the formation of blood clots . ITGA2 iAlthough the etiology of KD is elusive, a genome-wide association study (GWAS) showed that single-nucleotide polymorphisms (SNPs) of AGT, NEBL, ITPKS, TGF-β, and KCNN2 may be modify the occurrence of CAL in KD –25. To dCompared with the rs1126643 CC genotype, the harmful effect of the CT/TT variant genotype was more pronounced in children ≤60 months of age and in males. This finding is consistent with results of other studies . Our resThese results underscore the need to monitor the possibility of CAL in children with KD based on ITGA2 gene mutation. Early initiation of interventions will reduce the risk of mortality caused by CAL in children with KD.In summary, this study confirms that ITGA2 rs1126643 is associated with increased KD and coronary artery lesion susceptibility. However, future studies with larger sample sizes and practical experiments should be conducted to explore the roles of the ITGA2 gene in KD with coronary artery lesion susceptibility and severity.