We evaluated the association between endoscopic scores of colonic inflammation and fecal calprotectin (FC), fecal immunochemical occult blood test (FIT), and C-reactive protein (CRP) in patients with ulcerative colitis (UC). Endoscopic scores reflecting the most severe lesion [maximum Mayo Endoscopic Subscore (M-MES) and Ulcerative Colitis Endoscopic Index of Severity (UCEIS)] and those reflecting the inflammation of the entire colon [sum of MES (S-MES) and Ulcerative Colitis Colonoscopic Index of Severity (UCCIS)] were evaluated. FC, FIT, and CRP were measured, and their association with the four endoscopic scores was evaluated. Endoscopic scores of 78 complete colonoscopies (66 UC patients) were evaluated using the three biomarkers. FC and CRP tended to correlate more strongly with S-MES and UCCIS than with M-MES and UCEIS. In the M-MES 0, 1 group, compared to CRP, FC and FIT showed stronger correlations with S-MES and UCCIS. Conversely, in the M-MES 2, 3 group, only CRP was significantly correlated with each descriptor. CRP more strongly reflects colon-wide mucosal inflammation than FC and allows reliable assessment of inflammation throughout the colon in active UC. Since the goal of current treatment for IBD, including UC, is to achieve mucosal healing, it is important to accurately evaluate the inflammatory state of the mucosa in UC2. While endoscopy can be used for direct evaluation of the inflammatory state of the intestinal mucosa in UC, biomarkers indirectly reflect the state of inflammation in the colonic mucosa and can be used as a substitute for colonoscopy. C-reactive protein (CRP) is a biomarker that reflects systemic inflammation, which has been evaluated for its usefulness as a marker of IBD activity9. Recent studies have also described the usefulness of biomarkers in fecal samples, such as fecal calprotectin (FC), and the fecal immunochemical occult blood test (FIT), and reported that these biomarkers more accurately reflect local inflammation of the colon in patients with UC20.Ulcerative colitis (UC) is an idiopathic chronic inflammatory bowel disease (IBD) with recurrent symptoms of remission and relapse22, which represent the scores of the most inflamed lesions in UC. In recent years, new scoring methods for evaluating the inflammatory state of the entire colon have been reported. Kawashima et al. reported the sum of the MES (S-MES), which is the sum of the MES in five colonic segments , and reported the usefulness of FC in comparison with this new scoring system23. Lobatón et al. also evaluated the usefulness of the modified MES (MMES)24. Similar to the S-MES, the MMES involves totaling the MES in five segments, multiplying that number by the number of evaluated segments, and dividing the derived number by the number of inflamed segments. The MMES has been clinically, biologically, and histologically correlated with disease activity in UC. The UCEIS is calculated by totaling three descriptors, while the Ulcerative Colitis Colonoscopic Index of Severity (UCCIS) is calculated by scoring four descriptors in five segments and applying the scores in the formula for UCCIS26. UCCIS is rarely used in clinical practice because of the complexity of scoring. However, scoring is performed by applying the sum of descriptors to the calculation formula, allowing the objective evaluation of the inflammatory state of the entire colon, which is highly useful in clinical research.However, endoscopic assessment is the most accurate way to assess the state of inflammation in UC. Common endoscopic scores for assessing UC activity are the Mayo Endoscopic Subscore (MES) and the Ulcerative Colitis Endoscopic Index of Severity (UCEIS)Few reports have shown an association between biomarkers and endoscopic scores for evaluating the entire colon, such as the S-MES and UCCIS. In this study, the usefulness of FC, FIT, and CRP as biomarkers for UC was examined using the maximum MES (M-MES) and UCEIS, which show the most severe UC lesions, and the S-MES and UCCIS, which show the degree of inflammation throughout the entire colon. If biomarkers can be confirmed to correlate with colon-wide inflammation in patients with UC, the information may enable the assessment of active disease without the need for colonoscopy, which is associated with an increased risk of perforation in active disease.Patients with UC who were treated at the Hamamatsu University School of Medicine between February 2019 and November 2020 were enrolled in this study. The diagnosis of UC was performed according to established diagnostic criteria, based on clinical, endoscopic, and histological criteria. Patients with IBD who were not diagnosed with UC but were diagnosed with conditions such as indeterminate colitis or unclassified IBD were excluded. UC patients with a history of colorectal surgery were excluded because S-MES and UCCIS require the observation of the total colon. In addition, patients with acute infectious enterocolitis or regular intake of aspirin and/or other nonsteroidal anti-inflammatory drugs were excluded. Seventy-eight patients with UC who met the above criteria were registered.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.This was a prospective cross-sectional study. The purpose of this study was to assess the usefulness of FC, FIT, and CRP as biomarkers for UC by correlating these biomarkers with four endoscopic scores. The primary endpoint of this study was the identification of markers that show a significant correlation with each endoscopic score. The secondary endpoint was the correlation between the three biomarkers and each endoscopic score, divided into two groups .21. S-MES was calculated by totaling the MES in five colonic segments , as described above23.Patients with UC underwent bowel preparation consisting of the ingestion of a polyethylene glycol-based electrolyte solution prior to colonoscopy. UC endoscopic scores were assessed using the M-MES, S-MES, UCIES, and UCCIS. M-MES was graded as follows: 0, normal or inactive disease; 1, mild disease with erythema, decreased vascular pattern, and mild friability; 2, moderate disease with marked erythema, absence of vascular patterns, friability, and erosions; and 3, severe disease with spontaneous bleeding and ulceration in the lesion with the most severe inflammation22. The UCCIS score was assessed using the following descriptors in the fives segments, as in the S-MES: vascular pattern (score 0–2), granularity (score 0–2), erosions and ulcers (score 0–4), and bleeding/friability (score 0–2). These descriptor scores were then applied to the following formula: UCCIS = 3.1 × Sum (vascular pattern across five segments) + 3.6 × Sum (granularity across five segments) + 3.5 × Sum (ulceration across five segments) + 2.5 × Sum (bleeding/friability across five segments)26. M-MES 0 or 1 was defined as indicative of mucosal healing.The UCEIS score was calculated as the sum of three descriptors: vascular pattern (score 0–2), erosions and ulcers (score 0–3), and bleeding (score 0–3)27. CAI was evaluated using the following criteria: the presence of diarrhea (number of stools per day), nocturnal diarrhea, visible blood in stool (percentage of movements), fecal incontinence, abdominal pain or cramping, general well-being, abdominal tenderness, and a need for anti-diarrheal drugs27. Clinical remission was defined as a CAI ≤ 3.Clinical disease activity was evaluated using Lichtiger’s clinical activity index (CAI)Fecal samples were prepared on or before the day of colonoscopic preparation. Samples were collected in plastic tubes for FC measurement and shipped at − 20 °C, as recommended by the laboratory . FC was measured with a Phadia 250 immunoanalyzer  using the Elia A Calprotectin 2 reagent , via fluorescence enzyme immunoassay principles.To prevent bleeding due to the endoscopic examination, fecal samples were obtained on or before the day of colonoscopic preparation. A collection kit  was used to collect stool specimens. Submitted samples were immediately processed and examined using the OC Sensor io .According to routine clinical practice, serum CRP level was measured to assess the UC activity status of patients. Blood samples were collected within a few days of endoscopic examination. This measurement was performed at the Laboratory Test Department of Hamamatsu University School of Medicine.P < 0.05 was considered statistically significant.Statistical analyses of the data were performed using SPSS version 24 , SAS version 9.4 , and EZR  software. Correlation analyses were performed using Spearman’s rank correlation test. The study protocol was reviewed and approved by the ethics committee of Hamamatsu University School of Medicine (20-322). Further, the investigation was conducted in accordance with Good Clinical Practice principles and in adherence to the Declaration of Helsinki. All patients provided informed consent prior to enrollment in this study.Seventy-eight colonoscopies  were performed in patients with UC. The baseline characteristics of the patients are shown in Table Correlations between each of the three biomarkers and each of the four endoscopic scores were analyzed Table . Every eTo find the factors that were responsible for the correlations between the biomarkers and endoscopic scores Table , we evalWe divided all cases into a group with M-MES 0, 1, which is generally considered to reflect mucosal healing in UC, and a group with M-MES 2, 3, which is associated with endoscopic UC activity. In these two groups, the correlations of S-MES and UCCIS with each biomarker were analyzed Table . In the The correlations between the sum of the descriptors in UCCIS and the three biomarkers were evaluated in the M-MES 0,1 group Table . FC and In this study, we analyzed the usefulness of FC, FIT, and CRP as biomarkers for UC diagnosis using scores representing the most severe lesion (M-MES and UCEIS) and scores representing inflammation throughout the colon (S-MES and UCCIS). FC and CRP tended to correlate more strongly with S-MES and UCCIS than with M-MES and UCEIS. In the M-MES 0, 1 group, compared to CRP, FC and FIT showed stronger correlations with S-MES and UCCIS. Conversely, in the M-MES 2, 3 group, only CRP correlated significantly with each descriptor.22. There are reports of endoscopic scores that evaluate inflammation throughout the colon. In many of these reports, the colon was divided into five to seven segments, the state of the mucosa was evaluated in each segment, and the score of the entire colon was obtained26. However, such scoring systems for evaluating the entire colon are rarely used in clinical practice due to the complexity of evaluation. Endoscopic scores, such as the MES and UCEIS, which are widely used in clinical practice, indicate scores in the areas with the most severe inflammation, whereas biomarkers for UC, which have been reported to be useful in recent years, are considered to reflect inflammation throughout the colon. It is considered important to compare inflammation throughout the colon with biomarkers to evaluate the characteristics of UC biomarkers more accurately.To date, several endoscopic scores of UC have been reported, but the MES is the most widely used score in clinical practice and trials. In addition, the UCEIS is often used as an endoscopic score for UC. However, these scores present the lesion with the most severe inflammation and do not indicate inflammation in the entire colon23. In the study by Lobatón et al., the correlation coefficients of FC with MMES, which is synonymous with S-MES, and M-MES, were r = 0.669 and r = 0.725, respectively24. The correlations between various markers and cumulative MES (cMES), which is calculated by totaling the MESs of six segments , were analyzed in a previous study20. In that study, the correlation coefficients of FC with M-MES and cMES were r = 0.38 and r = 0.52, respectively, which are similar to our results.The four endoscopic scores showed a strong significant correlation with the biomarkers, particularly with FC and FIT. Although CRP showed a less strong correlation than FC and FIT, it showed a significant correlation with the four endoscopic scores. FC and CRP showed a stronger correlation with S-MES and UCCIS, which represent the presence of inflammation in the entire colon, than with M-MES and UCEIS, which indicate the most severe lesion. Kawashima et al. reported that the correlation coefficients between FC and M-MES or S-MES were r = 0.79 and r = 0.86, respectively, indicating that FC may more strongly reflect inflammation throughout the colon28. In their study, compared to FC, FIT was more strongly correlated with bleeding in the UCEIS and reflected the association of bleeding with inflammation in UC.Few studies have evaluated the relationship between the entire colon and various biomarkers. In the present study, FIT tended to have a larger coefficient of correlation with M-MES and UCEIS than with S-MES and UCCIS. Although the cause of these findings is unknown, it may be related to the ability of FIT to reflect the most severely inflamed lesion. Further case accumulation and analysis are therefore required. Furthermore, to examine the factors that reflect the inflammation of each marker, the descriptors of UCEIS and UCCIS were compared with each biomarker. The results of this analysis showed that FIT was strongly correlated with bleeding in the UCEIS and the sum of bleeding/friability in the UCCIS. Naganuma et al. reported similar results to those of the present study29. In their report, although there was a significant difference in FC values between groups with MES 0 versus MES 1 versus MES 2 in UC patients with extensive colitis, no significant difference was observed between the MES 2 and MES 3 groups. Contrarily, although no significant difference was shown in CRP value between the MES 0 and MES 1 groups, a significant difference was shown in the MES 1 versus MES 2 versus MES 3 groups. The difference between their study and ours is that they did not evaluate the endoscopic score of the entire colon; however, consistent with our findings, the authors found that CRP was more useful in the endoscopic active phase. In addition, as shown in supplemental Table Next, we examined whether the characteristics of biomarkers differ, depending on the presence or absence of endoscopic activity, using widely used endoscopic scores. In this study, mucosal healing was defined as M-MES 0, 1, and the correlations between endoscopic scores and biomarkers were evaluated. In the M-MES 0, 1 group, although FC, FIT, and CRP showed significant correlations with S-MES and UCCIS, the coefficient of correlation of CRP was lower than those of FC and FIT. Notably, in the M-MES 2, 3 group, only CRP showed a significant correlation with S-MES and UCCIS. In other words, from the viewpoint of the use of biomarkers for the evaluation of the inflammation of the entire colon, although it is desirable to use FC or FIT in the mucosal healing phase, evaluation using CRP may be more useful than using other biomarkers in the endoscopic active phase. Similar findings were also shown in a study by Sonoyama et al. that compared FC and CRP with endoscopic scoresFurthermore, to investigate the characteristics of the biomarkers in both the MES 0, 1, and MES 2, 3 groups, the correlation between the sum of descriptors in the UCCIS and each biomarker was analyzed. As expected, FC and FIT showed significant correlations with almost all items in the MES 0, 1 group, whereas these biomarkers did not show correlation with nearly all the descriptors in the MES 2, 3 groups. Conversely, CRP did not show significant correlations with each sum of descriptors in the MES 0, 1 group, whereas a significant correlation of each sum of descriptors was found in the MES 2, 3 group in all items, except for the sum of bleeding/friability. The results of this sub-group analysis reveal that CRP does not reflect the amount of bleeding in the endoscopic active phase but strongly reflects the presence of other factors.This study had several limitations. First, the sample size was small. In addition, the number of patients with active UC was small. In this study, the entire colon was evaluated in order to sum the MES and UCCIS. However, in clinical practice, the high severity of UC limits the number of cases in which total colonoscopy can be performed, due to the risks of patient distress and complications, such as perforation and bleeding. For this reason, the number of active UC cases was small in this study. However, even with the small number of cases, the significant correlation of CRP with the S-MES and UCCIS in UC patients in the endoscopic active phase indicates that CRP is more strongly correlated with inflammation throughout the colon.31. Therefore, we considered that CRP is an adequate marker of active colon inflammation, reducing the requirement of colonoscopy, with the associated risk.Patients with IBD have a higher risk of perforation than healthy individuals, and colonoscopy, particularly in patients with severe IBD, has the potential of further increasing the risk of perforationIn conclusion, CRP more strongly reflects inflammation throughout the colon than FC and FIT in UC patients with severe endoscopic activity. CRP is useful in situations where endoscopy cannot be performed .Supplementary Information.


Subclinical mastitis (SCM) is an economically important disease for dairy cattle worldwide; therefore, regular screening is imperative to detect SCM at an early stage so as to control it. The study was conducted to compare the test characteristics of sodium lauryl sulfate (SLS) as a test reagent to detect SCM in dairy cows.First, 106 milk samples of dairy cows were subjected to available indirect screening tests  considering somatic cell count (SCC) as gold standard test. Then 273 milk samples were allowed to react with different concentrations of SLS with or without sodium hydroxide (NaOH) and indicators (bromothymol blue [BTB] and bromocresol purple [BCP]).SLS with or without NaOH yielded best reaction with the milk samples similar to Leucocytest. It was observed that the reaction of milk samples with SLS added with indicators (BTB and BCP) was easier to visualize than without indicators. SLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP had high sensitivity, specificity, and substantial agreement with SCC. The area under the receiver operating characteristics curve of SLS 2% with BCP and SLS 3%+NaOH 1.5% with BTB was 0.917 and 0.875, respectively.It may be concluded that SLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP may be the potential reagents for the development of an effective cow-side test to detect SCM, as the main ingredient SLS is considerably cheap and readily available in developing countries. Mastitis is one of the most prevalent and costly diseases of dairy cows . It occuSe) and specificity (Sp) are lower compared to CMT  and bromocresol purple [BCP]).An equal volume (1 mL) of SLS solutions and milk sample was mixed on a shallow paddle having four cups, and the mixing was properly done by the gentle circular motion of the paddle for few seconds. The reaction developed almost immediately with milk containing a high concentration of somatic cells. The peak of reaction was obtained within 10 s and any change in the milk, such as viscosity, consistency was observed as mentioned below.Se, Sp, positive predictive value (PPV), negative predictive value (NPV), and kappa-value were calculated. The results were weighted on the basis of kappa value, which ranges from 1 to –1, where a value of –1 indicates complete disagreement between tests, 0 indicates agreement by chance only, 0.01-0.20 indicates slight agreement, 0.21-0.40 indicates a fair amount of agreement, 0.41-0.60 indicates moderate agreement, 0.61-0.80 indicates substantial agreement, and 0.81-1 indicates almost perfect agreement [SCC was considered as the gold standard test for the determination of epidemiological test characteristics of different indirect screening tests as well as test anionic surfactant - SLS. Five quality parameters: greement . The SPSSe against 1-Sp, that is, a ROC curve was generated and the performance of a diagnostic test was quantified by calculating the area under the ROC curve (AUROC). The ideal test would have an AUROC of 1.A graph of Se (64.9%) than that of WST (45.9%), Immucell (56.8%), and SFMT (63.9%)  along with SCC as gold standard test were employed on 106 milk samples of dairy cows to diagnose SCM and to compare the test characteristics. Leucocytest had comparatively higher  (63.9%) . Kappa-vSe, Sp and kappa-value of SLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP were higher than other concentrations of SLS  for both the tests was 0.917, which was higher than AUROC of other concentrations of SLS  was easier to visualize than without indicators. s of SLS . The Se s of SLS . The AURSe and Sp are intrinsic values of a test and are useful for comparing one diagnostic test with another [Se and Sp can be calculated only if there is an objective, independent means of determining whether the disease is truly present [SCM is not a problem of individual cows rather a problem at the dairy herd level; therefore, it is essential to test the dairy cows of the herd periodically for the screening of SCM . Se and  another . However another . Se and  present .Se and Sp taking SCC as the gold standard for the detection of SCM in dairy cows. SLS with or without NaOH at different concentrations gave better reaction with the milk samples similar to existing indirect screening test, Leucocytest. SLS decreases surface tension, change the structure and conductivity of cell membrane and nucleus, interfere osmotic balance, block oxidization and stimulate proteolytic enzymes, and increases milk viscosity. NaOH degrades somatic cells; as a result, the DNA of the cells reacts with the solution and forms gel, which indicates the presence of SCM. Earlier, SLS was evaluated to be used as CMT substitute; however, the test properties and interpretation of results were not clearly explained [Se of a test indicates the proportion of total infected quarters that are truly infected. The Se of SLS solutions was very much close and similar to the Leucocytest. It was also observed that the Sp of the SLS solutions was also high, which means that it is able to detect uninfected samples which are not truly infected. These highly satisfactory intrinsic values of the SLS solutions increased the acceptability of this solution to be used as a test solution to detect SCM in dairy cows.Here, an anionic surfactant, SLS was evaluated based on the xplained ,29. The Se, Sp and kappa-value of SLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP were higher than other concentrations of SLS. Among the available screening tests, the Se was very low in case of WST and SFMT. Muhammad et al. [et al. [Se of WST and SFMT. Although Leucocytest had high Se and Sp it is somewhat costly as well as not readily available whenever needed in our country. On the other hand, WST and SFMT are not appreciated due to its low Se.In addition, PPV and NPV were also high, which indicates that SLS identified milk samples from truly affected quarters as positive and from truly not-affected quarters as negative. Further evaluation was done by adding indicators to SLS to observe the reaction of milk. Indicator is a halochromic chemical compound added in small amounts to a solution so that the reaction of the solution can be determined visually ,37. Therd et al. , Sarker d et al. , and Bad [et al.  also repSLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP may be the potential reagents to be used as test solutions to detect SCM in dairy cows as SLS is available and relatively cheap in our country. However, field evaluation of these reagents is needed to validate their characteristics as a screening test to detect SCM in dairy cows.NST and MTI: Conceptualization. NST, MSI, and MK: Sample collection, data collection, and data curation. NST, MSI, MK, and MSP: Formal analysis. MTI: Funding acquisition. NST: Investigation. NST: Methodology. MTI and MAE: Project administration. NST, MSP, and MTI: Resources. MTI and MAE: Supervision. NST, MSP, and MTI: Validation and visualization. NST: Writing – original draft. NST, MSI, MK, MSP, MAE, and MTI: Writing – review and editing. All authors read and approved the final manuscript.


Streptococcus pneumoniae (Sp). Urine samples were collected at the initial visit, 24 h post-enrollment, and 10–14 days post-enrollment. Of the 122 children enrolled, 24% had a virus detected and 19.7% had Sp detected. Intraclass correlation coefficients demonstrated greater within-subject versus between-subject variability for all metabolites detected. In linear mixed models adjusted for age, time, history of asthma, Sp, and viruses, 1-methylnicotinamide was increased by 50% in children with Sp and decreased by 35% in children with rhinovirus/enterovirus. Children with Sp had 83% higher levels of trimethylamine-N-oxide compared with those without Sp. However, when adjusting for multiple comparisons, the association was no longer statistically significant. In conclusion, there appear to be short-term changes within the urinary metabolome of healthy children, but levels of metabolites did not statistically differ in children with viral carriage or Sp detected.The human metabolome may vary based on age, over time, and in the presence of viral carriage and bacterial colonization—a common scenario in children. We used nuclear magnetic resonance spectroscopy to identify and quantify urinary metabolites of children without signs or symptoms of respiratory illness. A urine sample and two nasopharyngeal swabs were collected to test for respiratory viral pathogens and colonization by  The human metabolome can be affected by environmental  and biological  factors. In adults, metabolites that differentiate sex are mostly the result of changes in energy metabolism , as men have more muscle mass than women ,2. In prStreptococcus pneumoniae (Sp) [Sp can become pathogenic, and cause significant morbidity, such as pneumonia [Sp, viral carriage, and their influence on metabolomes in asymptomatic children is essential. The nasopharynx of healthy children is likely to be colonized with commensal bacteria, such as iae (Sp) . In up tneumonia ,9. ChildSp colonization and viral carriage in the nasopharynx of healthy children without respiratory signs or symptoms. The urinary metabolome of children is ideal for studying metabolic changes, viral carriage, and bacterial colonization over a short time, as it is easily accessible and provides a comprehensive view of the child’s physiology. The objectives of this study were to (1) determine within-subject and between-subject variation in the urinary metabolomes of children over a short time period, and (2) to identify urinary metabolites associated with The study included 122 children who provided three urine samples, and had complete data available, out of a total of 185 enrolled. Most of the children  were between 5 and 12 years of age, and 11 (9%) had a history of asthma . No cigaStreptococcus pneumoniae (Sp) in their nasopharynx, determined by the presence of Sp autolysin A (lytA) . The most frequently detected virus in the nasopharynx was rhinovirus/enterovirus , with parainfluenza and coronavirus each detected in 3 (10.3%) of the children.Although all of the children were asymptomatic, 24 (19.7%) had a detectable load of A (lytA) . Among td-glucose) had greater than 90% missing values across samples and were excluded from the analysis. We compared the metabolite levels across the three timepoints . Metabolites that were statistically associated with Sp included 1-methylnicotinamide and TMAO. Only 1-methylnicotinamide was associated with the presence of rhinovirus/enterovirus. However, these associations did not survive multiple comparison adjustment. The low ICC values for all metabolites suggested a high within-person variance, indicating a low reliability or less stable fluctuations across time within the same child. Therefore, a single measurement in time may not be appropriate in children when investigating a response to therapy but may be useful in determining disease versus health at a single timepoint. We found that among a cohort of healthy children with no signs or symptoms of respiratory illness, 24% had a virus detected and 19.7% had Bacteroidetes and Ruminococcus [Bacteroidetes, and becomes less diverse with increasing age, which may also explain the increased levels of TMAO in older children compared with younger children [Older age was associated with six metabolites, two of which were tryptophan—an amino acid—and quinolinate—a metabolite of tryptophan—which commonly fluctuate due to diet . TMAO canococcus . The pedchildren . Cis-acochildren . Unlike children . Additiochildren ,14. In aSp detection in children. In Sp-infected mice, 20 metabolites were found to be associated with infection, specifically related to the TCA-cycle , gut microbiome , and others  [Sp detection, the concentration of TMAO increased in children with Sp colonization, in contrast to the decrease found in mice with Sp infection. The same study also found that 1-methylnicotinamide, which we found was associated with Sp detection, was also associated with methicillin-resistant Staphylococcus aureus. However, we did not test for MRSA, and are thus unable to verify this result in children. We also found that 1-methylnicotinamide may be associated with the presence of rhinovirus/enterovirus after adjusting for other clinical factors, but this association was not statistically different when compared with the null model. This metabolite is part of the nicotinamide pathway and has anti-inflammatory properties. Another study found that lower levels of 1-methylnictoniamide are present in children with asthma [We found that only two metabolites—TMAO and 1-methylnicotinamide—were potentially associated with nelline) . Although asthma . HoweverThere are several limitations to this study: First, we standardized all metabolites by urine creatinine concentration, as is common practice for urinary metabolomics analyses. Previous literature suggests that adjusting for creatinine could artificially create correlation with age . HoweverIn summary, our study highlights the importance of considering the overall age group in years across a cohort of children that are known to be colonized with bacteria or carry viruses. Here, we demonstrate that despite verified bacterial and viral colonization, the urinary metabolome was not systematically affected by these factors. Furthermore, we observed that the metabolome in children is highly variable within subjects, especially within the youngest age group, necessitating the inclusion of repeated measurements in future studies when investigating long-term changes , in order to adequately quantify the noise and distinguish it from true signal. This was a longitudinal study of healthy children ranging in age from 3 months to 12 years old. Children and their guardians  were recruited through electronic mail and social media outlets associated with the Schubert Research Clinic at the Cincinnati Children’s Hospital Medical Center. Recruitment occurred from April 2017 to February 2018 to capture children during one respiratory and one non-respiratory season. Children were excluded from the study if they were hospitalized 14 days prior to their visit, were immunodeficient or immunosuppressed, had a chronic pulmonary disease (except for asthma), chronic cardiac disease, sickle cell disease, neuromuscular disorder, or any history of aspiration or aspiration pneumonia.Participants received USD 20 as compensation for completing all of the study procedures. All guardians gave their informed consent for inclusion of their child, and assent was obtained in children >11 years, before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved as research on human subjects by the Ethics Committee of Cincinnati Children’s Hospital Medical Center (Protocol #2014-1390)Demographics and clinical data were prospectively obtained from the parent or legal guardian of the child. Demographics included age, sex, race, ethnicity, mother’s education, breast feeding for children <2 years old, and smoking status of the parents or legal guardians, along with the child’s exposure to smoke from other sources . Clinical data included history of asthma, history of pneumonia, prescription or over-the-counter medications the child was currently taking, immunization status , seasonal flu vaccination status, any current respiratory symptoms , and whether any medical care visits were sought within the timeframe of the study, along with the reason for the visit. We did not obtain data on muscle mass, height, or weight, and are therefore unable to comment on the effects of BMI or muscle mass on the metabolome. The clinical data were obtained to ensure that the children were healthy and had no respiratory symptoms at the time of urine sample collection. Additionally, data were collected on the inclusion of children from the same family, although no more than two children from the same family were eligible for inclusion.Urine samples were collected at three timepoints from each participant: at the initial enrollment, 24 h post-enrollment, and 10–14 days post-enrollment. For the initial enrollment sample, older children were given urine cups directly for collection. For younger children, cotton balls were placed in the child’s diaper. Urine-soaked cotton balls were then placed in a syringe provided by the study, and urine was extracted into a urine cup for further processing and storage. Prior to enrollment, we analyzed the cotton balls, urine collection cup, and syringe used via NMR. We found that the cotton balls produced negligible levels of glycerol and acetone. The urine collection cup and syringe had no metabolite contaminates. We found no age-related association with glycerol and acetone, and therefore did not exclude any samples from the younger children. Participants were given a home urine collection kit during enrollment, which included specific instructions regarding the collection and dropping off or mailing of the second and third urine samples, two pre-coated urine cups, two ice packets, an insulated mailing box, a shipping label, and two temperature strips to determine the amount of time for which the sample was at any temperature above 4 °C. Urine samples were collected at a single timepoint during the day, and were not pooled samples over the 24-h period. Urine samples were collected in urine collection cups coated with 10% sodium azide solution in order to prevent additional bacterial growth . Once urChlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila. All viral testing was qualitative; therefore, only the presence or absence of the viruses was reported. The NP swab in STGG medium was tested for Sp autolysin A (lytA), a gene fragment that indicates the presence of Sp [Sp load was quantitative; therefore, a value for each specimen was reported unless no Sp load was detected. We examined the load both as a continuous variable, and with the variable dichotomized as present (>500 copies/mL) or absent (≤500 copies/mL), based on the level of detection of the assay.At the time of enrollment, two nasopharyngeal (NP) swabs were also collected. NP swabs were placed in viral transport medium and skimmed milk–tryptone–glucose–glycerin (STGG) , and thece of Sp . Sp load2O was added to each sample as an internal standard. A one-dimensional 1H-NMR spectrum of each urine sample was acquired using the first increment of the standard NOESY pulse sequence on a Bruker 600 MHz spectrometer at the Cincinnati Children’s Hospital Medical Center [1H-NMR spectra were processed using Chenomx software  [At the time of the assay, samples were thawed at room temperature. Samples were randomly batched and processed to minimize artificial effects that may arise due to day-to-day variation in NMR runs. The pH of each sample was adjusted with NaOH or HCl to achieve a pH of 7.0 ± 0.25 . A voluml Center ,21. All l Center ,23. The  Canada) ,24.Sp presence, viral detection, and age–time interaction by including these variables as fixed effects in the linear mixed models. To account for multiple testing for the multiple metabolite outcomes, we used the Benjamini–Hochberg false discovery rate procedure [Continuous variables are presented as median and IQR, and categorical variables are presented as counts and percentages. The quantified metabolite data were log transformed to achieve a normal distribution of the data to conduct parametric statistical analysis ,25. Onlyrocedure . All tesrocedure . ICC androcedure ,31.


N = 77, mean age = 5.57) and adults . The goals of this study were to (1) examine the developmental trajectory of expression recognition and (2) examine the development of an OAB. Participants were asked to find a target face displaying an emotional expression among eight neutral faces. Results showed that children and adults found happy faces significantly faster than angry and fearful faces regardless of it being present on the faces of peers or non-peers. Adults responded faster to the faces of peers regardless of the expression. Furthermore, while children detected angry faces significantly faster compared to fearful ones, we found no such difference in adults. In contrast, adults detected all expressions significantly faster when they appeared on the faces of other adults compared to the faces of children. In sum, we found evidence for development in detecting facial expressions and also an age-dependent increase in OAB. We suggest that the happy face could have an advantage in visual processing due to its importance in social situations and its overall higher frequency compared to other emotional expressions. Although we only found some evidence on the OAB, using peer or non-peer faces should be a theoretical consideration of future research because the same emotion displayed on non-peers’ compared to peers’ faces may have different implications and meanings to the perceiver.People seem to differ in their visual search performance involving emotionally expressive faces when these expressions are seen on faces of others close to their age (peers) compared to faces of non-peers, known as the own-age bias (OAB). This study sought to compare search advantages in angry and happy faces detected on faces of adults and children on a pool of children ( A growing number of studies  point ouFrom an evolutionary point of view, prioritized detection for emotional cues on the faces of others was likely adaptive throughout human evolution as it helped survival by, for instance, foreshadowing danger (angry expression), or promoting trustworthiness (happy expression). Several research fronts are focused on the root causes of visual search advantage related to schematic faces , emotionIn parallel to the ASE, numerous other studies . FurtherMost of the aforementioned research in preschool children examining the advantage of emotional facial expressions in visual search tasks used mainly adult faces as targets . The perTherefore, our overarching goal of the present study was to test ASE vs. HSE on two independent samples of preschool children and adults, using photos of children as well as adult faces displaying emotions. Considering this age gap variable, the matrix of interactions comprises (a) a child observing another child’s facial expression, (b) a child observing an adult, (c) an adult observing another adult, and (d) an adult observing a child. When (a) a child observes another child’s angry face, anger might imply social rejection and even some danger as children might hurt each other. In contrast, when (b) an adult angry face is shown to a child, it may signal authority or a reprimand, for example, related to something necessary. However, it can also signal violence . In factDue to the versatility of the happy facial expression and its possible interpretations, happy faces are expected to present the highest search advantage in both adults and children, regardless of the age of the faces. Moreover, happy faces are also more frequently observed, leading to familiarity . In addithe target, was different from the others, i.e., the crowd. All pictures presented in one trial were of different people. In line with previous studies  as a within-subject factor and Group (adult or child) and Model (adult or child) as between-subject factors. The gender of models was balanced to present a female target in a female crowd in half of the trials, and a male target in a male crowd, in the other half.The control of low-level features is particularly necessary when studying a single group of participants responding to several types of emotion. If not well-controlled, one might erroneously find a misleading result, suggesting, for example, that particular emotion is more rapidly processed, when in fact, the angry pictures were a bit brighter by chance due to the eyes being more open or the teeth more showing. This study used a large number of pictures (70 adults and 80 children) controlled for color brightness, contrast, spatial frequency, and luminance values; exposed teeth ratio; and used two participant groups to minimize error.Our research was approved by the local Ethical Review Committee for Research in Psychology (Nr. 2018-62) and carried out following the Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed written consent was obtained from adult participants and parents of children participants, and oral consent was obtained from the children.1. The inclusion criterion was the successful completion of an emotion labeling task. First, participants were shown the faces used in the experiment and asked to name the emotions displayed on them. If they reached a success rate of 70%, a priori set by the authors, separately for each emotion, could progress to the visual search task2. If not, they were told that the experiment is over; children could choose a small gift for their participation. Including only those who could correctly identify the facial expressions was essential to reduce confounding biases and variance due to false recognition.The exclusion criteria for the current study were a history of depression, anxiety, or a neurodevelopmental disordern = 43) or (2) adults (n = 34), and (3) adults who completed the task with faces of children (n = 37) or (4) adults (n = 31). See details about the groups in the next two sections “Visual search with faces of children” and “Visual search with faces of adults”). The sample size for our study was determined by computing the estimated statistical power based on the effect sizes of prior experiments on HSE and ASE using a similar task , indicating that a total sample size of 12 would provide sufficient statistical power. A more conservative estimation  indicated that a total sample size of 76 is required. Therefore, we collected nearly double the required sample size. The post hoc analysis showed that the achieved power in this study was 0.99.A total of 146 volunteers participated in our study (78 children and 68 adults); about half of them completed the visual search task with faces of children, while the other half completed the visual search task with faces of adults. This design resulted in a total of four groups: (1) children who completed the visual search task seeing faces of children (lar task . For thisoftware . The inia priori criteria (70% success rate) and, therefore, were excluded. Forty-three  preschool children completed the visual search task. Their mean age was 5.65 years (SD = 0.78). Their ability to name the facial expressions displayed on the photographs used was tested (see section “Procedure”). The mean success rate for this sample was 91.71% (SD = 21.50%). The mean success rate for the whole sample was 72.88% (SD = 33.63%).Initially, 56 children were recruited. However, 12 children failed to reach a priori criteria of naming the facial expressions . All of the participants were Caucasian, right-handed, with normal to corrected-to-normal vision.The adult sample comprised 37  adults with a mean age of 21.8 years (SD = 0.78). All of the adults passed the a priori criteria (70% success rate) and were, therefore, excluded. The children tested sample comprised 34 (14 boys and 20 girls) preschool children who completed the visual search task. Their mean age was 5.47 years (SD = 0.75). The mean success rate of naming the facial expressions for this sample was 90.57% (SD = 20.90%). The mean success rate for the whole sample was 71.23% (SD = 34.68%).Initially, we recruited 44 children. However, 10 children failed to reach a priori criteria of naming the facial expressions . All of the participants were Caucasian right-handed with normal or corrected-to-normal vision.The adult sample comprised 31  adults with a mean age of 21.1 years (SD = 1.31). All of the adults passed the 2 < 1, p > 0.1).All of the pictures (target and crowd) were taken from the Dartmouth Database of Children’s Faces . The Dar2 < 1, p > 0.1). Again, photographs with neutral expressions served as the crowd.All of the pictures (target and crowd) were taken from the Karolinska Directed Emotional Faces database . The datThe 3 × 3 sets were created in a block arrangement , with eight crowd members (measuring 7.57° × 7.57° each), and one target (same size as background pictures). Images were separated with a 2pt wide black border. All three target emotions were presented in each of the nine possible locations, separately for boy and girl models. Thus, the stimuli set consisted of 54 matrices, i.e., nine trials of each of the six conditions. The male and female trials were presented separately, in 27-trial blocks, counterbalanced over participants, i.e., half of the respondents started with male trials, the other half started with female trials.Fs < 1, p > 0.1).Although visual search paradigms can be sensitive to potential low-level confounds, face color and original appearance were retained for ecological validity. We calculated color brightness, contrast, spatial frequency, and luminance values for each matrix using Matlab after data collection to monitor whether these values could be a source of bias. We found no significant difference between the calculated low-level visual feature values within the pictures. Furthermore, these values had no covariate effects on the results  completed the same procedure. Those who passed the emotion labeling task were taught how to use the touchscreen monitor if it was necessary. In the case of children, the experimenter helped them to create a drawing of their right hand on a sheet of paper. For adults, we used a previously printed and laminated paper that had the outline of a hand on it. Participants were asked to place their right hand on this paper between trials. Children were seated approximately 30 cm and adults approximately 60 cm in front of the monitor. First, all participants completed nine practice trial matrices, one with each target. Responses to practice trials were excluded from further analyses. If the experimenter saw that they understood the task, and the participants also gave their oral consent to continue, the experiment started. Respondents completed the task in two sessions with a short break between them. The instruction was to find the picture that shows a different emotional expression from the others as quickly as possible. For children, the experimenter started each stimulus by hitting a button on the keyboard; for adults, the images automatically appeared . Each image was preceded by a cartoon figure (for children) or a fixation cross (for adults) presented for 500 ms. Then, the participant indicated the target’s location by touching it on the touchscreen monitor. Upon completing the experiment, children could choose a small gift as a reward for their efforts. The task lasted for approximately 15–20 min, including the break between the two blocksTrials with very low pointing accuracy  or very high reaction time values (two standard deviations from the mean) were excluded from further analyses, comprising less than 1% of the data. The statistical analyses were performed using the JAMOVI Statistics Program Version 0.9. for Windows . ReactioF = 64.89, p < 0.001, and ηp2 = 0.31]. The Tukey corrected pairwise comparisons revealed that participants found happy faces  faster than angry  and fearful faces , but the latter two did not differ .The main effect of emotion was found significant . To tease apart this interaction, we used two separate ANOVAs for children and adults. These follow-up analyses revealed that, for adults, the main effect of emotion was significant  with the same pattern as reported above; i.e., participants found happy faces faster than angry  and fearful faces , while the latter two did not differ . For children, the main effect was also significant . However, the pattern was different: Children found happy faces faster than angry  and fearful faces  and also angry faces were found faster than fearful ones . See We also found a two-way interaction between Emotion and Group  and between Emotion and Model and Group .The other interactions involving Emotion were non-significant, i.e., between Emotion and Model , meaning that adults  were generally faster than children  in both tasks. Furthermore, the main effect of the Model was also significant  showing that participants were faster to identify the emotions on faces of adults  compared to faces of children .The main effect of the Group was significant , revealing that the main effect of the Model was only significant for adults but not for children. Interestingly, children detected emotions on the faces of adults  and faces of children  at similar speeds. In contrast, adults were faster to detect emotions on the faces of adults  compared to faces of children .The two-way interaction between Group and Model was significant [The present study sought to test visual search advantages for angry and happy faces, i.e., to compare ASE with HSE theories, respectively. Past research has shown mixed results, and an important possible confounding variable—namely the OAB—has been overlooked in previous research designs, as children usually completed the visual search for emotional expressions on the faces of adults. However, the emotions could portray very different meanings based on who sees it (a child or an adult) on whose face (a peer or a nonpeer). For example, previous research  has showOverall, our findings support the HSE across both samples. That is, faces displaying a happy expression were found quicker in a visual search task and had an advantage in visual processing corroborating previous studies  using adAdults also found happy faces first; nonetheless, we only found a marginally significant effect showing they reacted to angry faces slower than fearful faces regardless of the age of the model. It is likely that adults did not find anger on a child’s face as threatening . PresumaIn children, anger was detected faster than the fearful expression in line with our expectations. When anger is present on an adult’s face, it could signal reprimand or even aggression, triggering a quick defensive reaction . When itWe also found that adults detected emotions in other adults’ compared to children’s faces faster, an effect we did not observe in children. On the one hand, this could lend further support to the notion that the OAB is likely not the product of familiarity as older adults have necessarily previously been members of other age groups . Adult fSome limitations of this study shall be noted. First, we used only three emotional expressions based on previous studies investigating CFE and HSE. Nonetheless, including all basic emotions would be necessary for future studies, particularly neutral faces as controls. The visual search paradigm adopted in our study made it impossible to include a neutral condition. Prioritization of a specific emotion may be task-dependent see also . Also, tDespite these limitations, to our knowledge, this is the first study to demonstrate the happiness advantage effect in both children and adults using peer’s and non-peer’s faces. That is, young children found happy faces quicker compared to angry and fearful ones. This novel developmental evidence might add further support to the robustness and reliability of the HSE. Overall, this finding contributes to the understanding of the differences in detecting emotional faces. Using peer or non-peer faces should be a theoretical consideration of future studies because results will change based on the choice as emotions seen on either peer or non-peer faces have different implications and meanings to the perceiver.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Hungarian Ethical Review Committee for Research in Psychology. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AZ, VI, JB, and AS: conceptualization. AZ, NA, OI, BL, and CC: methodology. AZ, NA, OI, VI, and JB: formal analysis and investigation. AZ, NA, VI, JB, TM-B, OI, AS, BL, and CC: writing—original draft preparation. AZ, NA, VI, JB, TM-B, OI, AS, BL, and CC: writing—review and editing. AZ, NA, TM-B, OI, BL, and CC: funding acquisition. NA, VI, JB, TM-B, and OI: resources. AZ, AS, BL, and CC: supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Globally, concerns have been raised that the priority implementation of public health measures in response to COVID-19 may have unintended negative impacts on a variety of other health and wellbeing factors, including violence. This study examined the impact of COVID-19 response measures on changes in violence against women and children (VAWC) service utilisation across European countries.A rapid assessment design was used to compile data including a survey distributed across WHO Europe Healthy Cities Networks and Violence Injury Prevention Focal Points in WHO European Region member states, and a scoping review of media reports, journal articles, and reports. Searches were conducted in English and Russian and covered the period between 1 January 2020 and 17 September 2020. Data extracted included: country; violence type; service sector; and change in service utilisation during COVID-19. All data pertained to the period during which COVID-19 related public health measures were implemented compared to a period before restrictions were in place.Overall, findings suggested that there was a median reported increase in VAWC service utilisation of approximately 20% during the COVID-19 pandemic. Crucially, however, change in service utilisation differed across sectors. After categorising each estimate as reflecting an increase or decrease in VAWC service utilisation, there was a significant association between sector and change in service utilisation; the majority of NGO estimates (95.1%) showed an increase in utilisation, compared to 58.2% of law enforcement estimates and 42.9% of health and social care estimates.The variation across sectors in changes in VAWC service utilisation has important implications for policymakers in the event of ongoing and future restrictions related to COVID-19, and more generally during other times of prolonged presence in the home. The increased global attention on VAWC during the pandemic should be used to drive forward the agenda on prevention, increase access to services, and implement better data collection mechanisms to ensure the momentum and increased focus on VAWC during the pandemic is not wasted.The online version contains supplementary material available at 10.1186/s12889-022-12644-9. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 disease, was identified in Wuhan, China in December 2019, and by March 2020 had spread to over 100 countries and been declared a global pandemic by the World Health Organization (WHO) [Whilst such measures were intended to reduce the spread of COVID-19, concerns were raised about the potential for restrictions on movements to negatively impact other aspects of health and wellbeing. A particular area of concern was the impact of the restrictions on levels of violence and crucially, the ability of organisations to prevent and respond to violence. Whilst violence in public spaces was predicted to decrease as a result of stay-at-home orders and the closure of night-time economies, non-governmental organisations (NGOs) and other stakeholders working in the field of violence prevention warned of the risk of a ‘shadow pandemic’, where such measures compounded existing violence and abuse in private settings, such as the home, and exacerbated risk factors for already vulnerable individuals, particularly women and children –7.Globally, prior to the pandemic, over 1 billion children experience sexual, physical, or psychological abuse each year, whilst one in three women will experience intimate partner violence and/or sexual assault in their lifetime , 9. SomeWithout population-level surveys it is difficult to obtain current rates of VAWC across countries, and even more difficult to assess how levels of VAWC may have changed during the pandemic . Whilst Due to the time-sensitive nature of identifying the initial impact of COVID-19 on VAWC, the current study used a rapid assessment design to compile data on changes in service provision across WHO European Region. The rapid assessment used both primary and secondary data collection methods including a survey distributed across WHO European Healthy Cities Networks  and VioThe survey was distributed via the WHO Regional Office for Europe on behalf of the research team to individuals who are official focal points for different strands of WHO work, representing either: co-ordinators from a city member of the WHO EHCN; and/or WHO technical focal points for violence and injury prevention . The survey was available in English, Italian, and Russian, and asked participants to provide information and data on the impact of COVID-19 on service use related to VAWC. Informed consent was collected from all participants. Thirty-seven survey responses were received from individuals in the following member states: Austria, Azerbaijan, Bosnia and Herzegovina, Croatia, Czechia, Denmark, Finland, France, Georgia, Greece, Iceland, Italy, Luxembourg, Montenegro, North Macedonia, Norway, Portugal, the Russian Federation, Serbia, Spain, and the United Kingdom.Searches were conducted in English and Russian and covered the period between 1 January and 17 September 2020. Searches were conducted by two researchers (one for the English language searches and one for Russian language searches), with results exported into Excel and duplicates removed. English language searches were conducted in the International Newsstream of ProQuest , OVID , and Google Scholar. Russian language searches were conducted in East View Information Service, Yandex.ru, Radio Liberty’s Central Asian branches, Sputniknews, and Google Scholar (including eLibrary.ru and CyberLenika). Grey literature searches of relevant national and international organisational websites were also conducted in English and Russian. All references retrieved were subjected to an include/exclude process based on the full text. The inclusion criteria were that the publication contained data: related to VAWC service utilisation during the COVID-19 pandemic; related to one or more of the 53 Member States of the WHO European Region; and, suitable for extraction and synthesis . Three reviewers screened the full text to identify literature for inclusion, with 10% of data extracted by each reviewer (excluding data from the Russian language search) checked by a second reviewer.For each source, data was extracted on: country; income level of the country (using World Bank categorisation); violence type; service sector ; change in service utilisation during COVID-19; and, comparison period. In some instances, the same data was reported in more than one source. In such cases, only one change in service utilisation figure was extracted to reduce duplicate estimates, and all other extracted information was combined to increase the completeness of data. All service data were included regardless of the measure it related to . Website visit data was excluded as an increase in visits to websites does not necessarily reflect an increase in utilisation of the service. Service utilisation data were extracted as percentage change or calculated from raw data. All data pertained to the period during which COVID-19 related public health measures were implemented compared to a period before restrictions were in place and were country-specific. Percentage change was calculated by either comparing to the same period in 2019 (e.g. March 2020 vs. March 2019) or the same period before lockdown in 2020 (e.g. March 2020 vs. February 2020). In some cases, the comparison period used to calculate the percentage change was not specified.Data were analysed using SPSS (v.25). Analyses employed descriptive statistics to calculate frequencies and medians. Chi-square for independence was used for bivariate examination of associations between service sector and change in service utilisation (binary variable coding each estimate as increase or decrease in utilisation). To ensure the comparison period used to calculate the percentage change did not confound any relationship between change in service utilisation and sector, logistic regression was employed to examine the independent relationship between service sector and change in service utilisation, while controlling for the comparison period (including not specified). Modelled estimates for proportion of data points categorised as an increase in service demand were calculated for each sector using an estimated marginal means function to adjust estimates for comparison period . SensitiOverall, 350 data points related to VAWC service utilisation during our COVID-19 study period were identified from the survey and scoping review. Additional file Overall, across all three sectors, there was a 20.3% median increase in service utilisation (range: -9.6%—900.0%), with a median increase in service utilisation of 5.8% (range: -85.7%—300.0%) for law enforcement, and 47.5% (range: -85.0%—900.0%) for NGOs. Use of health and social services saw a median decrease of 8.0% (range: -89.6%—751.0%). The median percentage change in service utilisation across countries for each sector is presented in Figs. 2 = 69.76; p < 0.001). Most NGO estimates  reflected an increase in service utilisation, compared to 58.2% (n = 96) of law enforcement estimates, and 42.9% (n = 18) of health and social care estimates. In the multivariate analysis, after controlling for the type of comparison period used to calculate the percentage change in service utilisation during COVID-19, health and social service data was 17.23 (95% CI: 6.16–48.18) times as likely to show a decrease compared to NGO data, whilst law enforcement data was 9.69 (95% CI: 4.10–22.94) times as likely to show a decrease . Good data is critical to informing evidence-based policies and tailored programmes to respond to the needs of women and children during and after the pandemic, as well as during any future public health emergencies, conflicts, or natural disasters . For exaFindings in the current study should be interpreted in light of a number of considerations. Comparison periods used to calculate the percentage change in service utilisation varied across the collected estimates. However, we tried to mitigate the potential confounding of this factor by performing a multivariate analysis controlling for comparison period. Whilst data is presented and grouped based on the data source (e.g. police recorded data), comparisons should not be drawn between different countries. Differences between countries in, for example, legal definitions, recording practices, and data quality, mean figures are not directly comparable. Further, data may be related to different time periods or measures . As already discussed, much of the estimates were drawn from media sources which may introduce bias as these sources may be more likely to report ‘interesting’ or ‘shocking’ statistics and we had no way to check the accuracy of the reports. Whilst the study focused on VAWC service utilisation across the whole WHO European Region our searches were conducted in English and Russian and thus may have missed media reports in other languages. Further, whilst the survey requesting data from key stakeholders was sent to representatives across the whole WHO European Region, the quality of responses varied greatly.The COVID-19 pandemic may eventually wane, however, the impacts of the public health restrictions on other aspects of health and wellbeing may continue to affect individuals, communities, and societies in the long term. Findings from the current study suggest that during the pandemic, even fewer victims were reporting violence to the police or presenting at health services. However, NGO data suggested that violence had not reduced amongst these groups during lockdown, and service utilisation overall increased. National and international campaigns highlighting the impact of COVID-19 on the increased risk of VAWC in the home captured the attention of the global media and galvanised governments into action in many countries . GovernmThe authors alone are responsible for the views expressed in this publication and they do not necessarily represent the decisions or policies of the World Health Organization.Additional file 1.


This dramatic decrease in acute and oncological surgeries can most likely be ascribed to the fact that many patients avoided health care facilities because of the strict stay-at-home policy and/or the fear of contracting SARS-CoV-2 in the hospital. In view of future waves, the population should be encouraged to seek medical help for acute symptoms and to attend cancer screening programs.Innsbruck Medical University Hospital, Austria, provides the highest level of care for a region of approximately 1.8 million people. During the early COVID-19 outbreak in spring 2020 surgical activity was drastically reduced with the prime goal of preserving hospital capacities, especially intensive care beds. We conducted a retrospective analysis of surgical activities performed at Innsbruck Medical University Hospital during the lockdown period from March 15 to April 14, 2020 and compared these activities to the same period during the previous 5 years. Total surgical activity was reduced by 65.4% compared to the same period during the previous 5 years ( In accordance with these restrictions, all elective surgical procedures at Innsbruck Medical University Hospital were halted to reduce the risk of infection, harm and death from COVID-19 and to reserve personnel resources and medical equipment for the treatment of critically ill COVID-19 patients  were extracted from the surgical planning software myMedis . In a second step all surgical interventions were classified according to the organizational unit, date and time of surgery ; in a second step all surgical interventions were manually classified as elective, acute or oncology-related interventions by two medical doctors. The following interventions were classified as oncology-related surgeries: invasive diagnostics requiring anesthesia, major- and minor primary oncological surgery and follow-up procedures. Additionally, all patients were classified according to age, gender and American Society of Anaesthesiologists (ASA) physical status classification system.After receiving ethics approval  from the local Ethics Committee, data on all surgical interventions performed from March 15 to April 14 in the years 2015 to 2020 (t-test for continuous variables. We show effect size with estimated median differences for continuous data and odds ratios (OR) for binary variables, with 95% CIs.Statistical analyses were conducted using R, version 3.5.1. All statistical assessments were two-sided and a significance level of 5% was used. We present the absolute number of surgeries with a 95% CI  for the period 2015 to 2019, other categorical variables as frequencies (%) and continuous data as mean (95% CI). We applied the Exact Poisson test to assess the difference in the number of surgeries between 2015–2019 and 2020, Fisher's exact test for binary variables and the Welch two sample p = 0.0305; p < 0.0001; p < 0.0001; ASA 4: 5.3 vs. 2.7%; p < 0.0001; Age distribution of surgical patients was similar during the compared periods, whereas female gender was significantly more frequent in 2020 , 903 acute  and 241 oncological interventions . The largest reduction in surgical activity was seen at the Department of Orthopaedic Surgery . Surgical interventions performed during night shift hours were reduced by 36.4% (p < 0.0001). Analysis of the weekends (Saturday and Sunday) showed, that surgical activities across all departments were significantly reduced during the 2020 lockdown as compared to the previous 5 years , which is a reduction of 69% as compared to the previous 5 years (p < 0.0001).During the observed period in 2020 241 oncological surgeries were performed. Compared to the mean of the previous 5 years, this corresponds to a reduction of 47.8%  is most likely due to quarantine regulations in force at the time, which essentially prohibited all accident-prone outdoor activities such as skiing, mountaineering and paragliding. Additionally, with the closing of the Austrian border on March 15, about 150,000 tourists left the country, thereby significantly reducing the number of potential patients. Unexpected and therefore more difficult to explain, however, was the decline in acute interventions in other surgical disciplines, such as acute vascular occlusion in vascular surgery, acute appendicitis in general surgery or abscess incisions in oral and maxillofacial surgery. One possible explanation for the steep decline in acute interventions could be the so-called “COVID-19 collateral damage syndrome” (stay-at-home directive may have discouraged symptomatic patients from seeking early emergency treatment for non-COVID-19-related medical conditions; second, it can be hypothesized that also the fear of contracting COVID-19 in a health care facility may have further decreased the number of patients seeking medical help. This aspect is notable, especially in view of the potential collateral health damage that can occur, if certain medical conditions are not treated within a short timeframe (The reduction in trauma-associated acute surgeries (decrease of 67.6%; yndrome” . Many diyndrome” . The decimeframe . At Innsimeframe –12. Anotimeframe .Austrian mortality data show that between March 1 and April 30, 2020 overall mortality increased by 1%, peaking in mid-April, when mortality was 16% higher than in the same time period during the previous 5 years . HoweverIn summary, our data show that during the lockdown period from March 15 to April 14, 2020 acute and oncology patients were less likely to undergo surgical interventions by comparison to the same period in the previous 5 years. These findings emphasize once again a major challenge of the current pandemic, namely the difficult provision of access to health care facilities and medical services to the entire population, not only persons infected with SARS-CoV-2. On the one hand, this implies efforts to keep medical systems running, including cancer screening programs and diagnostic procedures; on the other hand, massive efforts should be undertaken in healthcare facilities to reassure patients that every possible precaution is being taken to prevent viral transmission, including rigorous and repeated testing of all healthcare workers and all admitted patients. In fact, since the end of May 2020 all surgical patients at the Innsbruck Medical University Hospital have been screened for SARS-CoV2 and since December 2020 all health care workers are obligated for weekly testing. It is of prime importance to create a healthcare environment where patients feel safe and are not afraid to seek medical help. If this is not ensured, the collateral damage from non-COVID-19-related health issues will cause enormous social and economic consequences for the entire healthcare system.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the Ethics Committee of the Medical University of Innsbruck, Austria.LG, GP, and JM: conceptualization, methodology, validation, writing the original draft preparation, supervision, and project administration. JA, LG, GP, and JM: formal analysis. JA and TH: data curation. JA, LG, GP, PS, SM, TH, and JM: writing the review and editing. TH: visualization. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Helicobacter pylori (H. pylori) infection impact tumor progression; however, the specific mechanisms remain controversial. We speculated whether this controversy is caused by differences in the location of TAM infiltration (in the core (CT) and invasive margin (MI) of primary tumors) and the topographical subsites of GC (cardia and non-cardia). Therefore, in this study, we investigated TAMs in different locations and H. pylori infection status as prognostic biomarkers for GC.In stage III gastric cancer (GC), the role of tumor-associated macrophages (TAMs) and H. pylori in 200 samples (100 cases of cardia-GC [CGC] and 100 cases of non-cardia GC [NCGC]) was performed. We compared the number of CD68+ and CD163+ macrophages that infiltrated the CT and MI in patients with the prognosis of CGC and NCGC, respectively. In addition, we analyzed the relationship between H. pylori status and the prognosis of patients with GC in different locations, as well as the correlation with TAM infiltration.Immunohistochemical staining for CD68 (pan-macrophage), CD163 (M2-like macrophage), and + macrophages that infiltrated the CT in CGC was associated with a better prognosis, whereas infiltration at the MI in NCGC indicated a poor prognosis. Furthermore, a high number of CD163+ macrophages infiltrating the MI resulted in a poor prognosis in CGC and NCGC cohorts. Considering the larger differences in the relationship between the infiltration of CD68+ macrophages at different locations and prognosis, we divided the GC cases into marginal and central GC, based on this difference. This resulted in an accurate estimation of the prognosis. Moreover, positive H. pylori status in central GC was significantly associated with a better prognosis and TAM infiltration.The distribution of TAMs had distinct characteristics in CGC and NCGC, especially differences between CT and MI subtype. A Kaplan–Meier analysis showed that a high number of CD68H. pylori status were identified as independent prognostic markers, with an obvious correlation between them. Therefore, it is important to clarify the impact of TAM location on the prognosis of patients with GC, which contributes to the development of potential therapeutic strategies.TAMs in different locations and  Gastric cancer (GC) is the fifth most common cancer worldwide, with nearly one million new diagnoses every year, and is the third leading cause of cancer-related deaths , 2. Most+CD8+ T cells by releasing CXCL9, 10, and 11 (Tumor-associated macrophages (TAMs) are macrophages that infiltrate tumor tissues and are among the most abundant immune cells in the tumor microenvironment (TME). Two main functional subtypes of macrophages have been described: M1 and M2. The function of M1-like macrophages include antigen presentation and tumor cell destruction, while M2-like macrophages promote extracellular matrix remodeling and angiogenesis and exhibit immunomodulatory characteristics . The ant, and 11 . Neverth, and 11  and the , and 11 . This co, and 11 . TherefoIn tumors, TAMs are usually stimulated by environmental factors to differentiate into M2-like macrophages. Due to the scavenging capabilities of M2 cells, the scavenger receptor CD163 has been proposed as a marker for M2-like macrophages. CD68 is considered the gold standard marker for human macrophages (Helicobacter pylori (H. pylori) is the most common chronic bacterial infection, affecting approximately 50% of the world’s population, and is a major risk factor for the development of GC  in most samples, while they mainly existed at the core (CT) in the NCGC group. The relationship between TAMs in different locations and the prognosis of patients with GC is conflicting. We thus utilized the difference to distinguish GC into central and marginal GC, to determine the relationship more accurately between macrophages and the prognosis of patients with GC. Moreover, a positive H. pylori status in central GC was significantly associated with better prognosis and TAM infiltrations. Taken together, these results suggest that TAMs and H. pylori are independent prognostic and predictive biomarkers for GC, and this finding might shed light on a new potential target for immunotherapeutic approaches for treating GC.In this study, we explored the relationship between TAM infiltration in different locations and the prognosis of patients with GC, and also whether TAM infiltration associates with This study retrospectively evaluated 200 stage III GC samples (100 cases of CGC and 100 cases of NCGC) from patients who underwent resection at the Tianjin Medical University Cancer Institute and Hospital between January 2012 and December 2014. None of the patients had received chemotherapy or radiotherapy before surgery. Patients with infectious diseases, autoimmune diseases or multiple primary cancers were excluded. All procedures were approved by the Ethics Committee of the Tianjin Medical University Cancer Institute and Hospital, and informed consent was obtained from all the subjects.H. pylori antibody . The next day, a secondary antibody was labeled with streptavidin–horseradish peroxidase and was applied to the sample, using a DAB staining kit (MXB Biotechnology).Paraffin-embedded slides were dewaxed in xylene and ethanol and steamed in a microwave oven with pH 9.0 ethylenediamine tetraacetic acid or pH 6.0 sodium citrate buffer to retrieve antigen epitopes. Once the samples had cooled, endogenous peroxidase activity was blocked using 3% hydrogen peroxide and the samples were blocked using goat serum. Slides were then incubated at 4°C overnight with the following primary antibodies: anti-human CD68 antibody , anti-human CD163 antibody , and anti-H. pylori status (negative or positive) was evaluated on the antral mucosa and corpora, which were stripped along the lesser curvature side. The degree of H. pylori infection was calculated by counting the number of bacteria (H. pylori) in each oil immersion field. The two pathologists randomly selected four fields of view to obtain the average value that was used to assess the relationship with the TAMs.The number and distribution of TAMs were evaluated to assess the role of TAMs in GC progression. For each tissue section, eight fields of view (four MI and four CT) were selected randomly for histological evaluation by two pathologists who were blinded to the clinical characteristics of the patients. First, CD68 and CD163 immunohistochemical staining was calculated from the number of positive cells, and the average number was recorded as the number of TAMs. Second, the geographic distributions of TAMs were evaluated to uncover the role of TAMs in different locations. Next, the 2 test was used to assess the relationship between clinicopathologic features of patients and CD68+ macrophages, CD163+ macrophages, and H. pylori infection status. Kaplan–Meier survival analysis was performed using ‘low’ or ‘high’ classifications according to the median number of CD68+ and CD163+ macrophages, and H. pylori status was based on the presence or absence of infection. Cox regression proportional hazard models were used to quantify hazard ratios for death from GC in both univariate and multivariate analyses. The models were adjusted for macrophages, H. pylori, age, sex, body mass index, carcinoembryonic antigen, tumor location, tumor size, and lymph node metastasis. The correlation between the number of TAMs and the degree of H. pylori infection was estimated using Spearman’s correlation analysis.IBM SPSS Statistics Software, version 20, was used for all statistical analyses. All p-values were two-sided, and the statistical significance cutoff was p ≤0.05. The χ+ and CD163+ macrophages at the MI in most samples was significantly higher than that in the CT  of promotes the M1 and M2 phenotypes, while a high MOI suppresses the M2 phenotype. Intriguingly, our current research focuses on a novel perspective that the degree of H. pylori infection relates to the CD68+ and CD163+ macrophage infiltration at different locations in GC tissue. However, the mechanism underlying this effect remains to be elucidated. Towards better understanding the underlying mechanism, and verifying the influence of H. pylori infection on the number, location, and polarization of tumor tissue infiltrating macrophages, an animal model of GC has been established. Furthermore, in vitro experiments are underway to determine whether H. pylori is itself a key factor. We hope that our future research will address these outstanding questions.The suppressive effect of he tumor , 38. It GC cells . Anothery et al.  proposedne cells . Indeed,rization , but alsastritis . In addiastritis  confirmeH. pylori and TAM infiltration on GC according to the topographic locations of tumors within the stomach. Our results suggest a new classification method based on CD68+ macrophage infiltration at the CT to evaluate the prognosis of patients more accurately with GC. We found that CD68+ macrophage infiltration in the CT and a positive H. pylori status were independent protective factors in central GC. These findings indicate that tumor location along with the location of infiltrating cells within the stomach should be considered when evaluating individualized patient prognosis. Furthermore, elucidating the detailed connection between H. pylori and TAMs will facilitate the development of new therapeutic strategies.In summary, our research differs from prior studies in that it focuses on the role of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. The patients/participants provided their written informed consent to participate in this study.QS and XR conceived and designed the experiments. ML, LZ, QZ, and YW performed the experiments. XR provided some suggestions. Data analysis was performed by YW and ML. Writing, reviewing, and manuscript editing was done by ML, QS, and XR. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Key R&D Program of China (2018YFC1313400), the National Natural Science Foundation of China , the Tianjin Natural Science Foundation (19JCYBJC27600 and 18JCQNJC81300), and the Scientific Research Program of Tianjin Education Commission (2019KJ185).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.