Objective To investigate depression and sexual function among pregnant and non-pregnant women throughout the COVID-19 pandemic.Methods A total of 188 women, 96 pregnant and 92 non-pregnant were included. The Beck Depression Inventory (BDI) and the Arizona Sexual Experience Scale (ASEX) were applied to the participants after obtaining sociodemographic data.Results The depression scores of pregnant and non-pregnant women were similar (p = 0.846). We found that the depression scores were significantly higher among the group of participants who have lower economic status (p = 0.046). Moreover, the depression score was significantly higher among women who lost their income during the pandemic (p = 0.027). The score on the ASEX was significantly higher, and sexual dysfunction was more prevalent among women who have lower levels of schooling and income (p < 0.05). Likewise, the ASEX scores were significantly higher (p = 0.019) among the group who experienced greater income loss throughout the pandemic. Upon comparing the pregnant and non-pregnant groups, we detected that sexual dysfunction had a significantly higher rate among pregnant women (p < 0.001).Conclusion In times of global crisis, such as the current pandemic, low-income families have an increased risk of experiencing depression and sexual dysfunction. When we compared pregnant women with non-pregnant women, depression scores were similar, but pregnant women were at a 6.2 times higher risk of developing sexual dysfunction. Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), coronavirus disease 2019 (COVID-19) began with the first suspicious cases in November 2019 in Wuhan, Hubei, China, in late December. After that, the disease shortly spread throughout the world, in about three months. It was declared a pandemic by the World Health Organization (WHO) on March 12, 2020, and the first case in Turkey was reported on March 11, 2020.Previous studiesThe female sexual response cycle can be divided into four phases: baseline, arousal (excitement), orgasm, and resolution. Regarding these phases, women might experience various sexual dysfunctions, such as lack of sexual drive and arousal, and inability to orgasm during sexual activity. It is an underestimated problem with a general prevalence between 20% and 50%.The present research aimed to investigate the depression and sexual functions of pregnant and non-pregnant women throughout the COVID-19 pandemic.The present is a cross-sectional trial conducted from May to August, 2020 with 96 healthy pregnant and 92 nonpregnant women, totalling a sample of 188 women. Most of the pregnant and non-pregnant women whom we invited to the study did not want to participate; they wanted to be in the hospital for a shorter time because they were afraid of contact transmission by the doctor. In total, 188 healthy women aged between 18 and 45 years who sought care at the Gynecology and Obstetrics Outpatient Clinic of a hospital in Turkey, and who did not have any known disorder, had not received medication for chronic conditions, and voluntarily agreed to participate in our study were included. Pregnant women who had fetal or maternal problems during their pregnancy, women who had a medical history of psychiatric or sexual dysfunction, and participants who submitted incomplete questionnaires were excluded from the study. The participants read and signed the informed consent form; after that, we collected sociodemographic data from them, and applied the Beck Depression Inventory (BDI) and Arizona Sexual Experience Scale (ASEX).Approval of the Ethics Committee was obtained (2020/514/176/9), with the project name of 2020–05–07T14_43_09, from the Ministry of Health of the Republic of Turkey to conduct the relevant scientific research.Sociodemographic data, such as age, level of schooling, occupational status, and obstetric status was obtained from the patients. They were asked whether they were pregnant or not, and, if they were, their gestational week was requested, and they were divided into three groups based on the trimester: first trimester , second trimester , and third trimester  groups. Based on data from May 2020 from the Turkish Statistical Institute, those with a total family monthly income below the hunger limit  are considered the lower-income class, those below the poverty line , the middle-income class, and those with a higher income , the upper-income class. Regarding loss of income during the pandemic, the participants were examined in 3 groups:, those who had no income loss, those who lost up to 50% of their income, and those who lost more than 50% of their income.The BDI was developed to measure the risk of depression, the level of the depressive symptoms, and the fluctuation in the severity of depression among adults. It is a self-administered questionnaire developed by Beck et al.The Arizona Sexual Experience Scale (ASEX), which has been developed by McGahuey et al.p-values were calculated using the Chi-squared test. For the continuous variables, thep-values were calculated using the Mann Whitney U test the and Kruskall Wallis test. Multivariable logistic regressions were used to calculate odds ratios (ORs) and 95% confidence intervals (95%CIs) for the associations between sexual dysfunction and depressive symptoms. Potential a priori confounders  were included in the models. All statistical analyses were performed using The Statistical Package for the Social Sciences  software, version 25.0.For the categorical values,The mean age of the sample was 30.1 ± 6.4 years. The demographics of the participants are presented inp = 0.046)  .p < 0.001). We also found that, as the level of economic income decreases, depression scores increase significantly (p = 0.009). The BDI score (mean = 13.9) was significantly higher among the participants who lost their income during the pandemic compared with those who did not experienced income loss (mean = 11.4) (p = 0.001). Furthermore, the BDI scores (mean = 14.0) of the patients whose relatives were diagnosed with COVID-19 was significantly higher than the scores of those whose relatives were not diagnosed (p = 0.013)  compared with those working in the public sector (mean = 9.5) ( 0.013) .p = 0.006). On the other hand, the percentage of moderate sexual dysfunction was significantly higher among multiparous women (p = 0.016). When the participants were categorized based on their depression status, moderate sexual dysfunction was determined to be high in patients with mild depressive symptoms, whereas the rate of severe sexual dysfunction was found to be high in those with severe depressive symptoms (p = 0.008)  .p = 0.005)  .When the factors affecting sexual dysfunction in women are analyzed by the multivariate analysis, odds ratio increase was found 1.18 times (95% CI 1.07–1.31), corresponding to each unit increase of age. Compared with those without depressive symptoms, the risk of sexual dysfunction in mildly-depressed patients increased 4.31 times (95%CI: 1.57 to 11.82), while this rate was of 5.57 (95%CI: 1.42 to 21.81) among participants with moderate/severe depression. The risk of sexual dysfunction increased 6.40 times (95%CI: 2.09 to 19.54) among unemployed patients compared with those employed in the private sector. Moreover, there was an increase of 6.20 in the OR (95% CI 2.15–17.90) among pregnant women compared with non-pregnant women .p < 0.001).When the correlation analysis was performed between the ASEX and BDI scores regarding the pregnancy status, a slightly positive significant correlation was found among pregnant women  higher among pregnant women. Low-income families and women have an increased risk of experiencing depression and sexual dysfunction in times of global crises, such as a pandemic.


Diagnosing COVID-19-associated pulmonary aspergillosis (CAPA) can be challenging since radiological and clinical criteria in the critically ill patient are nonspecific. Microbiological diagnostic support is therefore crucial. The aim of this study was to document the incidence of aspergillosis using bronchoalveolar lavage (BAL) as the diagnostic method and to determine the performance of the current mycological diagnostic tests most widely used for the diagnosis of CAPA, together with evaluation of the Asp lateral flow device (LFD).Prospective cohort study conducted between March 2020 and June 2022. Inclusion criteria were critically ill patients admitted to the ICU with SARS-CoV-2 pneumonia requiring invasive mechanical ventilation. Diagnostic bronchoscopy and BAL were performed at the beginning of invasive mechanical ventilation. The sensitivity, specificity, positive and negative predictive value (PPV and NPV), positive and negative likelihood ratio (LR + and LR-) of BAL culture, direct examination with calcofluor white stain, ELISA (Platelia) and LFD (AspLFD) for detection of galactomannan (GM) were evaluated. Aspergillus-qPCR was applied when discrepancies between diagnostic tests arose.Of the 244 critically ill patients with SARS-CoV-2 pneumonia admitted to the ICU, the majority  required invasive mechanical ventilation. Diagnostic bronchoscopic procedures were performed in 160 patients (80%), who were enrolled in this study. The incidence of CAPA was 18.7% (n = 30). LFD-GM demonstrated a sensitivity of 84%, specificity of 99%, PPV 94%, NPV 97%, LR(+) of 84, and LR(-) of 0.16. At GM-ELISA indices of ≥ 0.5 and ≥ 1.0, sensitivity was 92% and 79%, specificity was 95% and 99%, PPV 76% and 91%, NPV 99% and 96%, LR(+) 18 and 79, and LR(-) 0.08 and 0.21, respectively. The optimal cut-off index from the ROC curve was 0.48, with sensitivity of 95% and specificity of 95%.Using a diagnostic strategy based on bronchoscopy and BAL, we documented a high incidence of pulmonary aspergillosis in patients with severe SARS-CoV-2 pneumonia. Asp-LFD showed moderate sensitivity and excellent specificity, with a high PPV, and could be used for rapid diagnosis of patients with suspected CAPA. Recurrent coronavirus disease 2019 (COVID–19) pandemic outbreaks have placed a tremendous burden on healthcare, especially in patients admitted to the ICU requiring invasive mechanical ventilation –5. ThereWe hypothesize that invasive sampling is associated with increased diagnostic accuracy. We analyzed the diagnostic value of most of the latest microbiological techniques for the diagnosis of CAPA using invasive diagnostic methods. The aim of this study is to document the incidence of aspergillosis using bronchoalveolar lavage (BAL) as the diagnostic method, and to determine the performance of the mycological diagnostic tests most widely used for diagnosis of CAPA, together with evaluation of the Asp lateral flow device (LFD).This was a prospective observational study conducted in a major teaching hospital. Critically ill patients with SARS-CoV-2 pneumonia admitted to the ICU since the onset of the COVID-19 pandemic were included. The study period ran from March 2020 to June 2022. Patients were eligible if they were: (1) 18 years or older; (2) receiving invasive ventilation for COVID–19 SARS–CoV-2 infection confirmed by RT–PCR. Exclusion criteria were patients not requiring invasive mechanical ventilation, or in whom bronchoscopy was deemed unsafe. Diagnostic fibrobronchoscopy was performed in all mechanically ventilated patients on admission to the ICU or with suspected secondary infection. No patients received antifungal therapy prior first bronchoscopy bronchoalveolar lavage.Bronchoscopy and bronchoalveolar lavage (BAL) was performed with 150 ml of physiological saline solution, divided into three aliquots. The first 20 ml of the BAL was discarded, and a sample of the remaining fluid was collected for microbiological analysis. Fibrobronchoscopy was performed with disposable AMBU aScope™ 4 Broncho Regular OD 5.8 / 2.8 mm bronchoscopes . The bronchoscope was inserted using a special adapter valve to avoid generating aerosols during the procedure. A specialist pulmonologist with 30 years of experience not related to the research study randomly analyzed the BAL samples to perform a quality control; those with few alveolar macrophages and/or excessive numbers of airway-derived cells were considered unsuitable due to the low quality of the sample. No local anesthetics were administered, and aspiration was not attempted through the bronchoscope channel until it was in position at the site to be studied. BAL samples were sent to microbiology the service to be processed.Aspergillus genus.Qualitative galactomannan (GM) detection, culture on fungal media and calcofluor white staining were performed within the first hours, all tests were performed from the same BAL fluid. All BAL fluids were stored at -80ºC for quantitative GM detection and quantitative PCR (qPCR) assay for detection of the Aspergillus diagnostic GM antigen. The results were reported as weak positive or strong positive in less than one hour.The Aspergillus-specific lateral flow device  is a rapid immunochromatography test for the qualitative detection of BAL samples were processed according to the manufacturer’s recommendations . NeverthAspergillus species. Smear microscopy was performed using potassium hydroxide (KOH) and calcofluor white.Direct examination of BAL fluids by fluorescence microscopy used calcofluor white stain (Becton Dickinson) for microscopic observation of filamentous fungi. Staining was positive when we observed septate hyaline hyphae branching at 45 degrees, suggesting Aspergillus species were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and/or using colony characteristics and conidiophore and conidia morphology.BAL fluid was cultured on Sabouraud dextrose agar (SDA) with chloramphenicol (Becton Dickinson) prior to sample processing. For this, 10–20 ml of BAL was centrifuged for 10 min at 3000 rpm. 100 µl of sediment was inoculated onto fungal media. Fungal cultures were incubated at 30ºC for seven days. Aspergillus galactomannan assay  is an enzyme immunoassay (EIA) that detects the GM antigen produced by Aspergillus species during active growth. This GM-EIA was performed twice a week on BAL fluid from these patients. Two different cut-off points were considered to determine positive results: GM index ≥ 0.5 ng/ml and ≥ 1.0 ng/ml.The Platelia Aspergillus species ELITe MGB® Kit) was performed for the detection and quantification of the DNA of the Aspergillus genus: Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus flavus, Aspergillus versicolor and Aspergillus glaucus. Only copy numbers > 150 of the genus Aspergillus (Ct < 36) were considered positive.In cases where there were discrepancies because only one of the previously described tests was positive, a qPCR assay  Detection of GM by AspLFD and/or EIA and BAL culture-positive for Aspergillus species and positive Aspergillus-qPCR;2) Detection of GM by AspLFD and/or EIA and/or BAL culture-positive for Patients were defined as negative for CAPA when all the diagnostic tests returned negative results or only one test was positive.2 test or Fisher’s exact test.For the baseline characteristics of the study population, data were reported as absolute counts (%) or as medians [25th-75th percentile] and p values were assessed by Mann Whitney U test, Pearson’s χAspergillus galactomannan assay, culture on SDA with chloramphenicol and calcofluor white staining were analyzed.The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR(+)) and negative likelihood ratio (LR(-)) of AspLFD, Platelia Aspergillus galactomannan assay, the area under the curve (AUC), including 95% confidence intervals (CI), was examined to assess the ability of the diagnostic test to discriminate between patients with CAPA and critically ill patients without CAPA. The Youden index was applied to determine the optimal ROC curve cut-off value.In a receiver operating characteristic (ROC) curve analysis for the Platelia Aspergillus galactomannan assay at a cut-off index of ≥ 0.5 and ≥ 1.0. The strength of agreement was defined according to Altman’s scale as: < 0.20 (poor); 0.20 to 0.40 (fair); 0.40 to 0.60 (moderate); 0.60 to 0.80 (good); and 0.80 to 1.00 (very good agreement).Observed percentage agreement and the kappa statistic were used to measure agreement between AspLFD and the Platelia P value less than 0.05 was interpreted as statistically significant.Any The Statistical Package for Social Sciences Software  was used for statistical analysis. The local research ethics committees approved this study.A total of 244 patients with severe SARS-CoV-2 pneumonia were admitted to the ICU; 200 patients (82%) underwent mechanical ventilation, and diagnostic fiberoptic bronchoscopy was performed in 160 (80%) patients. During the study period, a total of 297 BAL fluid samples were collected; bronchoscopy was repeated in several patients during their ICU stay, indicated by clinical suspicion of ventilator-associated pneumonia. Finally, 285 BAL samples from 160 patients were selected for analysis  BAL samples tested positive. Culture on SAD with chloramphenicol was positive in 19/40 (47.5%) BAL samples, but only one with a positive calcofluor white stain. The GM-EIA (≥ 1) was positive in 30/38 (78.9%) BAL samples, and GM-EIA (≥ 0.5) in 35/38 (92.1%). Twenty-eight of 36 BAL samples (77.7%) showed positivity by both AspLFD and GM-EIA. All combinations of the positive diagnostic microbiological tests performed on BAL samples are shown in Fig. Aspergillus galactomannan assay (≤ 0.5) and culture on SDA with chloramphenicol. The false negative results were all positive by Aspergillus-qPCR. Five of these were found to have a BAL GM value > 0.5, and the only one at the GM index of ≤ 0.5 was BAL culture-positive for A. terreus complex.Sensitivity and specificity were 84% and 99%, respectively Table . There wAspergillus on SAD culture (A. terreus complex and A. flavus complex). Sensitivity was 9% and specificity 100%. Figure Only three cases were positive. In all cases, AspLFD was positive. Two patients had Aspergillus species isolated from fungal cultures in 13/30 patients (43.3%) with CAPA were: Aspergillus terreus complex in three patients (23%), Aspergillus niger complex in four (31%), Aspergillus flavus complex in four (31%) and Aspergillus fumigatus complex in two patients (15%). In 6 patients, different Aspergillus species were isolated  and were considered contaminations because all other tests were negative, including Aspergillus-qPCR. The median optical density index by Platelia Aspergillus galactomannan assay was 3.17 (2.95–3.88) from 19 determinations. Only one BAL sample with growth of Aspergillus terreus complex was negative for GM in both the AspLFD and the Platelia Aspergillus assay (GM = 0.49). This case was considered positive by Aspergillus-qPCR. The culture showed a sensitivity of 48% and a specificity of 98% . In these patients, Aspergillus-qPCR was positive.At a cut-off of 0.5, BAL sensitivity and specificity were 92% and 95%, respectively , with values between 1.0 and 1.3, were negative in all tests. Platelia GM showed 8 false negative cases (8/221), all with a positive /33, withThe optimal cut-off index for this assay in ROC curve analysis was 0.48, with 95% sensitivity and 95% specificity. The AUC of the ROC curve was 0.947 (95% CI: 0.882 to 0.997) . Positive percent agreement was 87% (28/32) and negative percent agreement was 92% (191/207).With an index ≥ 0.5, agreement between AspLFD and the Platelia Aspergillus galactomannan test showed substantial agreement, demonstrating good concordance (kappa coefficient = 0.79). Positive percent agreement was 81% (26/32) and negative percent agreement was 98% (202/207).At an optical density index ≥ 1.0, the comparative evaluation of AspLFD and the Platelia Diagnosing CAPA in the ICU presents a real challenge, not only because of the non-specificity of the clinical and radiological criteria, which are present in many other causes of respiratory deterioration in severe SARS CoV2 pneumonia, but also because of the heterogeneity of respiratory samples indicated for the diagnosis of aspergillosis.Cryptococcus species, Trichosporon species, and Fusarium species), antibiotics (e.g. tazobactam) and blood products, nutritional support, and even food taken during the hospitalization. Criteria for clinical suspicion of pulmonary aspergillosis associated with SARS-CoV-2 are defined as fever not explained by any other cause and/or impaired respiratory function, as measured by paO2/FiO2. The radiological criteria include the presence of pulmonary infiltrate on a simple chest x-ray. Since these are common criteria for SARS-CoV-2 infection itself, it is essential to base it on a microbiological diagnosis.Performance of fiberoptic bronchoscopy should also be promoted in order to obtain good quality invasive samples. It is glaringly obvious that an active search for this complication should be made , supportAspergillus-qPCR in BAL (< 36 cycles) are sufficient criteria to diagnose CAPA [Aspergillus-qPCR, and 6 patients with SDA culture of Aspergillus species returned negative results by AspLFD, the Platelia Aspergillus galactomannan assay and Aspergillus-qPCR.The diagnostic criteria for CAPA are based on the 2020 ECMM/ISHAM consensus criteria. Detection of fungal elements in BAL indicating a mold, positive BAL culture, BAL GM index ≥ 1.0 or BAL LFA index ≥ 1.0, and a single positive ose CAPA . The useose CAPA , 19. By Aspergillus spores present in the environment. Invasive samples are more reliable for the diagnosis of aspergillosis. As is well known, Aspergillus species are common colonizers of the respiratory tract, and their isolation in other samples such as tracheal aspirate culture could simply be colonization. In our study, only 19/34 (56%) patients had positive BAL cultures for Aspergillus species, although four Aspergillus samples were considered contaminated since they had negative Aspergillus-qPCR results and were negative by both GM-EIA and AspLFD. These results are lower than in another recent study, in which 22/33 (66.6%) patients were BAL culture-positive for Aspergillus species [Aspergillus species and so may be overestimated. In our study, the BAL culture yield was the third lowest of the mycological tests and only direct examination with calcofluor white staining had a lower performance. These results are similar to those reported in a cohort of patients with hematologic disease and invasive pulmonary aspergillosis, in which only 2/67 (3.6%) BAL fluid samples were positive in direct smears stained with calcofluor white [In critically ill patients admitted to the ICU with SARS-CoV-2 infection, CAPA is a serious complication that increases mortality . In thes species , althougor white . The invThis study has some limitations. This was a real clinical practice study conducted in a single center, and therefore requires further studies with the participation of more ICUs to confirm the findings. As this was a clinical study, the determination of galactomannan in serum was not taken into account, given that the patients were not neutropenic. It would be interesting to know what the outcome would be in a patient population with severe SARS-CoV-2 pneumonia, as this has not been studied previously. The main strength of this study in patients with severe SARS-CoV-2 pneumonia was the invasive diagnostic strategy used, despite the fact that, at the beginning of the pandemic, fibrobronchoscopy was not recommended by different societies for fear of generating aerosols. Since the beginning of the pandemic, BAL was performed after intubation in most patients. Another novelty is that we tested a diagnostic method like lateral flow in critically ill patients with suspected CAPA. This may be advantageous for early treatment of a disease associated with high mortality, in which the microbiological diagnosis often takes time to be confirmed.Using a diagnostic strategy based on bronchoscopy and BAL, we documented a high incidence of pulmonary aspergillosis in severe SARS-CoV-2 pneumonia patients. This is the first study to analyze AspLFD as a mycological marker for the diagnosis of CAPA. The sensitivity was not very high, but its positive predictive value was 94%, which could be helpful for rapid diagnosis of this disease. A delay in the diagnosis of CAPA is associated with a poor prognosis and increased mortality. In addition, the combination of several mycological markers improved the diagnosis of CAPA by reducing the false positives associated with diagnostic tests.


Veillonella spp. are obligate, anaerobic, Gram-negative bacteria found in the human oral cavity and gut. Recent studies have indicated that gut Veillonella promote human homeostasis by producing beneficial metabolites, specifically short-chain fatty acids (SCFAs), by lactate fermentation. The gut lumen is a dynamic environment with fluctuating nutrient levels, so the microbes present shifting growth rates and significant variations of gene expression. The current knowledge of lactate metabolism by Veillonella has focused on log phase growth. However, the gut microbes are mainly in the stationary phase. In this study, we investigated the transcriptomes and major metabolites of Veillonella dispar ATCC 17748T during growth from log to stationary phases with lactate as the main carbon source. Our results revealed that V. dispar reprogrammed its lactate metabolism during the stationary phase. Lactate catabolic activity and propionate production were significantly decreased during the early stationary phase but were partially restored during the stationary phase. The propionate/acetate production ratio was lowered from 1.5 during the log phase to 0.9 during the stationary phase. Pyruvate secretion was also greatly decreased during the stationary phase. Furthermore, we have demonstrated that the gene expression of V. dispar is reprogrammed during growth, as evidenced by the distinct transcriptomes present during the log, early stationary, and stationary phases. In particular, propionate metabolism (the propanediol pathway) was downregulated during the early stationary phase, which explains the decrease in propionate production during the stationary phase. The fluctuations in lactate fermentation during the stationary phase and the associated gene regulation expand our understanding of the metabolism of commensal anaerobes in changing environments.IMPORTANCE Short-chain fatty acids produced by gut commensal bacteria play an important role in human physiology. Gut Veillonella and the metabolites acetate and propionate, produced by lactate fermentation, are associated with human health. Most gut bacteria in humans are in the stationary phase. Lactate metabolism by Veillonella spp. during the stationary phase is poorly understood and was therefore the focus of the study. To this end, we used a commensal anaerobic bacterium and explored its short-chain fatty acid production and gene regulation in order to provide a better understanding of lactate metabolism dynamics during nutrient limitation. Veillonella spp. are obligate anaerobic, Gram-negative bacteria found in the human oral cavity  and in the colon , are linked to human health  medium with an initial optical density (O.D.) of 0.01 and was found to have a short lag phase. Sequentially, V. dispar underwent a log phase, then late log phase, early stationary phase, and stationary phase  analysis. During anaerobic growth, the lactate in the TYL medium was continually consumed, and the concentration was found to have been reduced to 48.5 mM, probably mostly d-lactate, at 21 h. Thus, V. dispar encounters l-lactate limitation at some point during the stationary phase. Acetate production was stable during early cultivation but significantly increased from 18 h to 21 h (stationary phase). Propionate production slowed down from 15 h onwards (-lactate . The TYL onwards . As a reV. dispar in the tryptone-yeast (TY) medium with an initial O.D. of 0.01 as a control. V. dispar grew poorly in the TY medium, with the O.D. only reaching 0.072 at 21 h. V. dispar cultivated in the TY medium produced about 1/50 of the propionate and 1/3 of the acetate compared to when it was grown in the TYL medium during late growth phase . Reacted cells during the log phase had low targeted intracellular metabolite levels compared to the other phases . The correlation coefficients for comparison pairs within the same biological group were generally >0.95, indicating that there was good biological reproducibility of the samples within the same group , and 201 genes were downregulated during the early stationary phase. On the other hand, 90 genes were upregulated and 375 genes were downregulated during the stationary phase . The genes upregulated between the early stationary phase and the stationary phase showed little overlap . These DEGs were then subjected to KEGG enrichment analysis, which revealed that propionate metabolism, gluconeogenesis, several amino acid metabolisms/biosyntheses, ribosome genes, and nitrogen metabolism were differentially expressed  at the transcript level used log phase as a control. Our results show that 149 genes were upregulated , pduCDE (propanediol dehydratase), and sucD , all of which were significantly downregulated during the early stationary phase  acts as an upstream regulator of acetate production and was significantly upregulated during the stationary phase. Its upregulation explains the increase in acetate production by V. dispar during the stationary phase (poxB (pyruvate dehydrogenase [quinone]) and ppsA (phosphoenolpyruvate synthase) were both significantly upregulated during the early stationary phase but downregulated during the stationary phase  with the downregulated genes, including trpC, trpE, aroA, aroB, aroC, aroE, aroF, aroK, and pheA; valine, leucine, and isoleucine biosynthesis genes were downregulated during the stationary phase  with the downregulated genes, including leuA, leuB, dmdA, tdcB, and ilvD; nitrogen metabolism genes were upregulated during the early stationary phase (Table S1) with the upregulated genes, including norB (nitric oxide reductase), glnA (glutamine synthetase), gltB (ferredoxin-dependent glutamate synthase), and narK (nitrite facilitator); biotin biosynthesis genes were significantly upregulated during the stationary phase (Table S1) with the upregulated genes, including bioB, bioD, bioF, bioK, and bioW; and finally, various ribosomal protein were downregulated during the early stationary and stationary phase (Table S1).In addition to propionate metabolism and gluconeogenesis, the gene involved in metabolism/biosynthesis of several amino acids, in nitrogen metabolism, in biotin biosynthesis, and a number of ribosomal proteins were also modulated during the various growth phases. Histidine biosynthesis genes were upregulated during the early stationary phase  with the upregulated genes, including V. dispar during various different growth phases, with a focus on the bacteria's gene expression and its major metabolites. In particular, the transition states between log phase and stationary phase, and between late log phase and early stationary phase, were studied. In the present study, V. dispar was cultivated in the TYL medium, which contains sodium dl-lactate as a carbon source. The growth of V. dispar in TYL medium using sodium l-lactate alone and sodium dl-lactate are indistinguishable. V. dispar was also cultivated in the TY medium as a control. V. dispar grew poorly in the TY medium and produced only a little propionate and about one-third of the acetate at 21 h compared to growth in TYL medium  during the log phase to producing a nearly equal amount of propionate and acetate (1:0.9) during the stationary phase. Acetate and propionate (both SCFAs) play important roles in human physiology, and therefore our results provide helpful information when the roles of Veillonella spp. in human physiology are being explored in the future. Specifically, the growth phase of the organisms within the human gut needs to be given seriously consideration.Microbes respond to nutrient limitation by reprogramming their metabolisms, such as by increasing fermentation or by decreasing aerobic respiration , 23. Ourry phase  and C. AL medium . Though L medium . The incL medium . Third, L medium  may be tog phase . Our traog phase . Althougxokinase , V. parvpyruvate , which sry phase , the uprV. dispar has an altered lactate metabolic profile during the stationary phase, and to further explore this the gene regulation of related metabolic pathways needs to be elucidated. Transcriptomics was used to investigate V. dispar gene expression during the log phase, early stationary phase, and stationary phase. Our results provide information on the transcript levels of almost all of the genes encoded by V. dispar and reveals that V. dispar exhibited a dynamic transcriptome during growth from log phase to stationary phase. Both hierarchical clustering analysis , citrate lyase, nitrogen metabolism, biotin biosynthesis, and amino acid biosynthesis. These findings agree with a previous study showing that E. coli is able to reshape its proteome during the stationary phase; these changes mostly involved differentially expressed proteins relating to metabolism : tryptone (BD Difco), 5.0; yeast extract (BD Difco), 3.0; sodium dl-lactate (Sigma), 12.0; K2HPO4 (Merck), 4.672; KH2PO4 (Sigma), 3.154. The pH value was adjusted to 7.0 at room temperature by NaOH. TY medium contains the same ingredients as TYL medium except for the absence of sodium dl-lactate and was used as a control medium for studying the V. dispar growth. All culture medium and buffers used in the following experiments were de-aerated by at least 72 h equilibration in an anaerobic chamber. Bacteria were cultivated in an anaerobic glove chamber . Bacterial growth was measured by monitoring the OD of the culture medium at 600 nm by using a spectrophotometer. The contamination by other organisms was excluded by observing colony morphology macroscopically and Gram staining microscopically.L medium , 42. TYLThe lactate and SCFAs in the medium were extracted using the protocol of a previous study . In brie(i) Resting cell preparation. An overnight culture of V. dispar was subcultured in a flask containing TYL medium, and the initial OD was adjusted to 0.01 ± 0.005. The bacterial culture (150 mL) was incubated statically at 37°C in an anaerobic incubator. V. dispar grown in TYL medium was sampled at different cultivation times by centrifugation , then the cells were washed twice. The washing buffer contained 75 mM potassium chloride, 75 mM sodium chloride (Sigma), and 2 mM magnesium chloride (Merck) in 2 mM potassium phosphate buffer . Bacterial cultivation, washing, and resuspending were all conducted in an anaerobic glove box.(ii) Lactate catabolism metabolic activity. The analysis of lactate catabolism metabolic activity was carried out as described previously (V. dispar harvested at different cultivation times were resuspended in 40 mM potassium phosphate buffer (pH 7.0), and the optical density of bacterial suspension was adjusted to OD 1.0 ± 0.05. The catabolic reaction was initiated by mixing 990 μL bacteria suspension and 10 μL of 1.6 M stock solution of sodium lactate to make the final concentration of sodium lactate of 16 mM. The reaction mixtures (1 mL) were incubated at 37°C in an anaerobic incubator for 60 min and terminated by adding 10 μL of 6 N perchloric acid. In brief, 16 μmol of sodium lactate (8 μmol of sodium l-lactate) was added into the reaction mixture, and the lactate consumption (μmole) and metabolites secretion (μmole) were analyzed. The reaction mixtures were centrifuged , and the supernatant was collected and filtered through a nylon membrane . The supernatant was then analyzed using a HPLC-PDA (photodiode array) system  to measure lactate consumption and the presence of various metabolic products . The pellet was further analyzed in order to measure its intracellular metabolite content.eviously , with so(iii) Intracellular metabolite analysis. The pellet after lactate catabolic reaction was collected and then washed twice with 0.85% saline solution. The intracellular metabolites were extracted using the cold methanol method . The supernatant was transferred to another clean tube. The extracted pellets were extracted again with 0.5 mL of methanol (–80°C). The intracellular metabolites of the pellets were dissolved in 1.5 mL of methanol. The samples were sent to the mass spectrometer center, National Yang Ming Chiao Tung University Instrumentation Resource Center for analysis using a LC-QTof system (Waters ACQUITY UPLC).l method . In brie(i) Plate counting. Samples of the bacterial cultures were collected, centrifuged , and then the supernatant was discarded. The pellet was resuspended in TYL medium and serially diluted to an appropriate concentration. Thereafter, 100 μL of the diluted bacterial suspension was evenly spread on an agar plate, which was followed by incubation in an anaerobic chamber for 48 h at 37°C. Plates with colony numbers between 30 and 300 were chosen for colony-forming unit (CFU) calculation. The final concentration of CFU/mL was calculated as the number of counted colonies multiplied by the number of times the bacterial suspension was diluted, and then multiplied by ten, as only 100 μl of bacterial suspension was added to the agar.(ii) Fluorescent staining. A LIVE/DEAD BacLight Bacterial Viability kit (Thermo Fisher Scientific) was used to determine cell viability. The cell viability assay followed the recommended protocol from the manufacturer. The 2× fluorescent dye working solution was prepared by mixing 2.5 mL component A (SYTO 9) and 2.5 mL component B (propidium iodine) according to the manufacturer’s instructions. This is optimized for live/dead cell contrast.g, 3 min, 4°C) and washed twice with water. For preparation of dead cells, bacteria in the log phase were harvested and washed with 75% ethanol. Cell pellets were resuspended in water by pipetting, and the cell concentration was adjusted to an optical density of 0.25. Next, 100 μL cell suspension and 100 μL fluorescent dye were mixed and incubated in the dark at 25°C for 15 min. For fluorescence microscopy, 5 μL final solution was transferred to a glass slide, covered with an 18-mm square coverslip, sealed with nail polish, and then imaged using an automated fluorescence microscope (Olympus BX63). SYTO 9 fluorescence was detected with the FITC filter cube; propidium iodine fluorescence was detected with the TRITC filter cube.Bacteria in the stationary phase (grown at 21 h) were harvested, centrifuged  and 6.5% solution B   was used in this study. The autosampler was set at 10°C. YMC-Triart C18  was used and protected by a guard column. To measure lactate, pyruvate, acetate, and propionate metabolic activity, the column oven temperature was set at 25°C, and the mobile phase consisted of 93.5% solution A  . The flog for 1 min, 25°C). Total RNA was extracted using a Presto mini RNA bacteria kit according to the manufacturer’s instructions. The concentration of each extracted RNA sample was determined by using a Qubit fluorometer. The purity and integrity were assessed using a NanoPhotometer and an Agilent FA5200, respectively. For the RNA sequencing, cDNA libraries were prepared using the Illumina Ribo-Zero Plus rRNA Depletion kit according to the manufacturer’s instructions. Illumina pair-end reads (150-bp) RNA-seq were performed on an MiSeq platform by HGT Co. (Taiwan). The reads were trimmed and filtered for data quality control using a Phred score of 30. RNA-seq reads were mapped against the V. dispar ATCC 17748T reference genome. The raw counts were normalized using the trimmed mean of M values (TMM) method. The normalized gene matrix was analyzed using iDEP  under the BioProject number


Background: Although the Bhanja bandavirus (BHAV) is widely distributed in some European countries, human infections are rarely reported. This study analyzed the prevalence of BHAV antibodies in patients with neuroinvasive diseases of unsolved etiology. Methods: A total of 254 Croatian patients who developed neurological symptoms during the four consecutive arbovirus transmission seasons (April 2017–October 2021) were tested. Cerebrospinal fluid (CSF) and urine samples were tested using RT-qPCR. In addition, CSF and serum samples were tested using a virus neutralization test. Results: BHAV RNA was not detected in any samples, while neutralizing (NT) antibodies were detected in serum samples of 53/20.8% of patients (95% CI = 16.0–26.3). In two patients, BHAV NT antibodies were detected in the CSF, indicating a recent infection. Both patients were inhabitants of rural areas in continental Croatia, and one reported a tick bite two weeks before symptoms onset. The seropositivity was high in all age groups (15.2–29.1%). The majority of seropositive patients (94.3%) resided at altitudes less than 200 m above sea level. The prevalence rates correlated positively with population density and negatively with certain climate parameters . Conclusions: The presented results indicate that BHAV is distributed in Croatia. Further studies are needed to determine the clinical significance of this neglected arbovirus. Phenuiviridae, genus Bandavirus [Haemaphysalis intermedia tick collected from a paralyzed goat in Bhanjanagar, India [Bhanja bandavirus  is a tick-borne bunyavirus that belongs to the family ndavirus . The virr, India . A laborr, India .Atelerix albiventris), and African ground squirrels (Xerus erythropus) are the vertebrate hosts of BHAV. Adult animals usually do not show clinical signs; however, the virus is pathogenic for young ruminants causing fever and signs of central nervous system (CNS) involvement. Vectors of the BHAV include metastriate ixodid ticks of the genera Haemaphysalis, Dermacentor, Hyalomma, Rhipicephalus, Boophilus, and Amblyomma. Haemaphysalis ticks are the main vector species in Europe [Haemaphysalis were recorded in Europe and North Africa [Although the BHAV is widely distributed in central Europe, around the Mediterranean basin, across the Middle Eastern countries to India, and in Sub-Saharan Africa, human clinical cases are rarely recorded . Sheep, n Europe . Eight sh Africa , while sh Africa .Hemaphysalis punctata female ticks collected from sheep on Brač Island [Haemaphysalis punctata is adaptable to different climatic conditions and inhabits various habitats, ranging from cold to mild and humid to dry [In Croatia, BHAV was isolated in 1974 from č Island . It is aH. punctata ticks were also found [In 1974, there was also a report of the first human laboratory BHAV infection in Croatia, followed by two additional laboratory infections recorded in 1977 . After Bso found .Seroepidemiological investigations conducted after the virus isolation in Croatia found BHAV hemagglutination-inhibiting (HI) antibodies in 31.5% of the inhabitants of Brač Island (1975 and 1977) . In the However, more recent data on the BHAV prevalence in Croatia are missing. The aim of this study was to analyze the prevalence of BHAV antibodies in patients with neuroinvasive diseases of unsolved etiology who developed the symptoms during the arbovirus transmission season.This study included 254 patients with unsolved neuroinvasive infections  from continental and coastal Croatian regions who developed symptoms during the four consecutive arbovirus transmission seasons (April 2017–October 2021). The median patient’s age was 51 (IQR = 31–67) years. Cerebrospinal fluid (CSF) and serum samples were collected in all patients, while urine samples were available for 98 patients. Sampling was performed during the acute phase of the disease. Viral etiology of the neuroinvasive disease was suspected based on the CSF analysis .CSF and urine samples were tested for the presence of neuroinvasive arboviruses: tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Usutu virus (USUV), Toscana virus (TOSV), Tahyna orthobunyavirus (TAHV), and BHAV. Viral RNA was detected using a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR): TBEV  , WNV (TaFor BHAV RNA (RT-qPCR), a Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies) and Rotor-Gene Q real-time PCR cycler  were used. Viral RNA was extracted using a High Pure Viral Nucleic Acid Kit (Roche Applied Science). The primers targeting the 166 nt region of the C segment of the BHAV genome and probe used were the following: forward primer—GAT GGT TGC ATA CAC TGA, reverse primer—CTT GGC ATC ATC TTT CCA, and probe—FAM-ATC CTT AAG GAG TTC GGT GAG GA-BHQ-1. Reactions were performed in 20 µL reaction volume containing 5 µL RNA, 0.4 µM of each primer, 0.2 µM of probe, 10 µL of 2x Brilliant III Ultra-Fast QRT-PCR Master Mix, and 1 µL of RT/RNase block. The conditions were as follows: 10 min 50 °C, 3 min 95 °C, and 40 cycles of 10 s 95 °C, 10 s 54 °C, and 30 s 72 °C.Serum and CSF samples were tested for the presence of arboviral antibodies: TBEV/WNV/USUV , TOSV , TAHV, and BHAV  .50; tissue culture infectious dose 50) was calculated using the Reed and Muench formula [50 of BHAV were added and the mixtures were incubated for one hour at 37 °C with CO2. Finally, 50 µL of 2 × 105 Vero E6 cells/mL were added to each well. The plates were incubated at 37 °C with CO2 and, starting from the third day, the inoculated cells were inspected daily for the cytopathic effect. NT antibody titer was defined as the reciprocal value of the highest serum dilution that showed at least 50% neutralization. Virus back titration, negative, and low positive control were included in each run. An NT antibody titer of ≥10 was considered positive.BHAV strain IG690 grown in Vero E6 cells was used as an antigen for VNT. Prior to VNT, the antigen was titrated in Vero E6 cells. The virus titer .The differences in seropositivity rates according to patients’ demographic/clinical characteristics  were compared using a Chi-square test. The strength of the association between dependent (VNT positivity) and independent variables was assessed by logistic regression. The associations between prevalence rates and climate/meteorological data  were analyzed using linear regression (lm function) in R . ClimateBHAV RNA was not detected using RT-qPCR in any of the tested samples, while BHAV NT antibodies were detected in serum samples of 53 (20.8%) patients (95% CI = 16.0–26.3) with titers ranging from 10 to 40. In addition, antibodies were detected in the CSF of two patients, indicating a recent BHAV infection .Demographic and clinical characteristics and laboratory results of patients with neuroinvasive disease are presented in p < 0.001) according to clinical presentations with significantly higher prevalence rates in patients with meningitis (44.7%) and febrile headache (22.2%) than with meningoencephalitis and myelitis (10.0% each).The prevalence of BHAV NT antibodies according to demographic and clinical characteristics and risk analysis are presented in The geographic distribution of BHAV-seropositive patients is presented in p < 0.001). In counties with higher population density prevalence rate was higher. Data from the City of Zagreb were removed from the statistical analysis since the population density in this region is a few folds greater than in any other region, making it an outlier.BHAV prevalence rates differed among counties . The geop = 0.012). In patients residing at altitudes lower than 250 m, there was also a significant negative association between the prevalence rate and mean yearly temperature (p = 0.037), the average number of warm days , and hot days . Total precipitation amount (p = 0.401), number of cold days , or cumulative insolation duration (p = 0.254) showed no influence on prevalence rates (The vast majority of the BHAV NT antibody-positive samples (94.3%) were collected from patients living at lower altitudes. Only three positive samples were collected from patients living at an altitude higher than 200 m. On the other hand, a significant increase in the prevalence rate with higher altitudes in patients residing under 250 m a.s.l. was observed  was recorded in two patients. In one patient, antibodies were detected in the CSF, while the serum sample was negative. In some patients with arboviral neuroinvasive disease, it was observed that IgM in CSF could appear even earlier than in the serum , which cNovember . AlthougNovember .In addition, BHAV NT antibodies were detected in the serum samples of 20.8% of patients with neuroinvasive disease suggesting the BHAV circulation in Croatia. Since the control group of asymptomatic individuals was not tested it was not possible to compare the seroprevalence in patients with CNS infection with the seroprevalence in the general population, which is a limitation of this study that should be addressed. However, previous Croatian studies showed lower seroprevalence rates in the general population of north Croatia (5.53%), suggesting the potential role of BHAV in the etiology of neuroinvasive infections.Ostryo-Carpinion adriaticum, which may be found in the elevated center of Brač, does not appear on Hvar; b) H. punctata ticks have much lower population densities on Hvar than on Brač; and c) very few domestic ruminants graze on Hvar , in contrast to their large numbers on Brač [Only a few seroprevalence surveys on BHAV were conducted in Croatia. One study conducted on Brač Island  in 1975 showed a very high overall BHAV seropositivity rate of 31.54%. Seropositive individuals were detected in 8/19 places tested . In cont on Brač . The majFew studies analyze the BHAV seroprevalence in the general population of Europe, mainly in endemic regions, whereas data on the seroprevalence in patients with neuroinvasive disease are very scarce. In a study from Italy (1967–1968), the prevalence of HI antibodies in humans was 1.8% with the majority of seropositive individuals detected in south and central regions . One stuThe present study showed no significant differences in the BHAV NT antibody prevalence rates between age groups 15.2–29.1%). However, it was interesting that the highest seropositivity rates were observed in the youngest participants , which may suggest a recent seroconversion. In addition, the interesting finding was a U-shaped association between age and seropositivity rate , which is also seen in neuroborreliosis [.1%. HoweThe significant observation is a higher seroprevalence rate in patients with meningitis (44.7%) and febrile headaches (22.2%) compared with meningoencephalitis and myelitis (10.0% each), suggesting that BHAV disease is usually mild and the main presentation of the neuroinvasive disease is meningitis.H. punctata, the main vector of BHAV, was recorded on ten different host species, mostly from ungulates, as well as one carnivore and one bird [In Croatia, one bird . This spone bird ,33,34,35one bird ,37,38. Tone bird .H. punctata from Brač Island is the second tick species from which BHAV was isolated in 1977 [ in 1977 . Brač Is in 1977 .In the presented study, the majority of BHAV-seropositive patients resided at lower altitudes (less than 200 m a.s.l.). Still, in these regions, a significant increase in the prevalence with higher altitudes was observed. Vector-borne diseases are particularly sensitive to climate change since ectothermic arthropods have their internal temperature regulated by external environmental conditions. The extrinsic incubation period (replication of arboviruses within vectors) occurs faster at higher temperatures. Vector biting rates also tend to increase with temperature . BHAV veAdditionally, BHAV prevalence rates positively correlated with population density. More time spent outdoors, whether for recreation  or gathering various edible (asparagus) and medicinal plants , or fruits , and mushrooms, could be a contributing factor for higher seropositivity in counties with higher population densities. As a result, they are more frequently exposed to tick bites and tick-borne pathogens.The detection of 20.8% of seropositive Croatian patients suggests that BHAV infections frequently remain unrecognized. Prevalence rates differed between counties. The majority of BHAV-seropositive patients were detected in patients who resided in areas at lower altitudes (less than 200 m). However, the prevalence rates increased significantly with higher altitudes in patients from these areas. In patients residing at lower altitudes, there was also a significant negative association between the prevalence rate and temperature and the number of warm/hot days. In contrast, precipitation amount, number of cold days, or cumulative insolation duration showed no influence on BHAV prevalence. Due to the small number of participants, further studies are needed to determine the prevalence and clinical significance of this neglected arbovirus in the Croatian population.


Platelet-rich plasma (PRP) injection for ankle osteoarthritis (OA) treatment showed contradictory results. This review was aimed to pool individual studies which assessed the efficacy of PRP for ankle OA treatment.Q test and the I2-statistic were assessed. Standardised (SMD) or unstandardised mean difference (USMD) and 95% confidence interval (CI) were estimated and pooled across studies.This study was conducted following the preferred report items of systematic review and meta-analysis guideline. PubMed and Scopus were searched up to January 2023. Meta-analysis, or individual randomised controlled trial (RCT), or observational studies were included if they involved ankle OA with aged ≥ 18 years, compared before–after receiving PRP, or PRP with other treatments, and reported visual analog scale (VAS) or functional outcomes. Selection of eligible studies and data extraction were independently performed by two authors. Heterogeneity test using Cochrane . The average age was 50.8–59.3 years, and 25–60% of PRP injected cases were male. The number of primary ankle OA was accounted to 0–100%. When compared to before treatment, PRP significantly reduced VAS and functional score at 12 weeks with pooled USMD of − 2.80, 95% CI − 3.91, − 2.68; p < 0.001 , and pooled SMD of 1.73, 95% CI 1.37, 2.09; p < 0.001 , respectively.Three studies from meta-analysis and two individual studies were included, which consisted of one RCT and four before–after studies with 184 ankle OAs and 132 PRPPRP may beneficially improve pain and functional scores for ankle OA in a short-term period. Its magnitude of improvement seems to be similar to placebo effects from the previous RCT. A large-scale RCT with proper whole blood and PRP preparation processes is required to prove treatment effects.Trial registration PROSPERO number CRD42022297503.The online version contains supplementary material available at 10.1186/s13018-023-03828-z. Ankle osteoarthritis (OA) presents with progressive cartilage destruction and may affect walking, physical and functional abilities, and also quality of life. Its incidence has been systematically approximated at 3%, particularly in the young and female . AccordiVarious conservative managements  including PRP have been widely used –6. The eThe contradictory results among diverse populations, treatments, and level of evidences bring about a gap of knowledge. Therefore, this study questioned “Does the PRP improve pain and function for ankle OA when compared to other conservative treatments?”. This systematic review and meta-analysis were conducted to pool the effect of PRP on pain and functional scores in ankle OA patients. This study hypothesised that PRP administration for ankle OA would positively alleviate pain and promote function.Eligible criteria for individual studies identified from meta-analysis were individual studies within systematic reviews and meta-analysis either RCTs, non-RCTs, cohort study, before–after study, or case series; involving ankle OA in adults aged at least 18 years; comparing between PRP and other treatment or placebo or before–after receiving PRP; and had any of following outcomes including pain or functional score. Duplicate studies and studies with inadequate details for data extraction after failed contacting the corresponding authors for three times were excluded.Eligible criteria for individual studies other than meta-analysis after the index date were RCTs, non-RCTs, cohort study, before–after study, or case series; recruiting ankle OA in adults aged at least 18 years; reporting PRP and other treatment or placebo or before–after receiving PRP; and had at least one outcome of interest . Duplicate studies and studies with insufficient or inaccessible data after contacting the corresponding author for three attempts were excluded.PubMed and Scopus databases were searched from their inceptions to 31 January, 2023. The Preferred Reporting Items for Systematic review and Meta-Analysis (PRISMA) guideline was applied , and a rIndividual studies included in previous meta-analyses and individual studies that were additionally identified from the last recent index searching date were selected for eligibility by two reviewers . Titles and abstracts were screened, and then, full articles were retrieved. The eligibility studies were reviewed and selected according to inclusion and exclusion criteria. Reference lists of the relevant articles were also checked. Disagreement between the two reviewers was resolved by consensus with the third author.Two authors  independently extracted data using standardised data extraction forms. The forms comprised of study design, year of publication, number of patients, mean age, cause of ankle OA , severe ankle OA , functional scores by the American Orthopedic Foot and Ankle Society Score (AOFAS) , Ankle OAll outcomes were evaluated at 12 weeks with regard to the maximum treatment effect. Only minimally clinical important difference of AOS was available for ankle OA as 28/100 . PossiblIntra-articular PRP injection was the intervention of interest. Possible comparators were intra-articular injection of inactive interventions ), conservative treatment , active interventions , and surgery . Since PRP had various techniques, types of preparation, dosage, methods, and times of administration were taken into account. If there were not enough studies, the interventions were grouped as active and inactive comparators, in which active intervention was a combined PRP with mesenchymal stem cell (orthobiologic serum). Commercial brand lists of PRP  includinThe quality of the study was independently appraised by two authors using the Cochrane Collaboration’s tool for assessing risk of bias for randomised controlled trials (RoB 2)  for RCTsThe Grading of Recommendations Assessment, Development and Evaluation (GRADE)  was used2-statistic. A standardised (SMD) or unstandardised mean difference (USMD) and 95% confidence interval (CI) were estimated and pooled across studies using a random-effects model if heterogeneity was present ; otherwise, a fixed-effects model was applied. A meta-regression was applied to assess a source of heterogeneity by regressing the mean difference on platelet concentration and PRP volume. Sensitivity analysis was performed by excluding studies that might be a source of heterogeneity. Publication bias was assessed using Egger’s test [A direct meta-analysis was performed comparing pain and functional scores between before and after receiving PRP. Heterogeneity was evaluated using Cochrane Q test and the Ir’s test  and a fur’s test . In the r’s test .A two-stage network meta-analysis was applied if the number of studies was sufficient to pool effects of PRP relative to active and non-active comparators as follows: First, a relative intervention effect, i.e. SMD/USMD, and its variance–covariance were estimated for each study; second, these effect sizes were pooled across studies using a multivariate meta-analysis. All possible relative treatment effects were estimated. Probability of being the best intervention was approximated using surface under the cumulative ranking curves (SUCRA). The consistency assumption was assessed using a design-by-treatment interaction model. A publication bias was evaluated using a comparison-adjusted funnel plot. A cluster rank was constructed for simultaneously considering probability of pain or functional scores, and possible adverse events such as pain, allergic reaction, skin discolouration, blood clot, and infection.P-value < 0.05 was considered as non-statistical significance.All analyses were performed using STATA Program 17.0. After considering patients and intervention terms, 81 studies were identified from PubMed and Scopus databases  are shown in Table P < 0.001), Fig. Q = 82.91; P < 0.001, I2 96.38%). A meta-regression was applied to assess if platelet concentration and PRP volume contributed to VAS score changes. Only PRP volume, but not platelet concentration, was significantly associated with VAS mean difference. Every increment in PRC volume by one unit would significantly reduce VAS score by 0.39 , Additional file I2 of 97.45% and 93.01%, respectively  with I2 90.99%  and − 1.83 , Additional file P < 0.001) showed asymmetry. Consequently, the contour-enhanced funnel plot was constructed and suggested that asymmetry might be caused by heterogeneity and publication bias  with Q = 4.87, P = 0.18; I2 = 38.44% when compared with before treatment  nor funnel plot showed asymmetry for functional score  and low leucocytes (< 1000 cells/mm3) after filtration are mandatory for the optimal outcomes [PRP preparation forms might also effect on the treatment outcomes. Meta-regression of 4 included studies –13, 29 soutcomes , 29.3 [3 [3 and leucocyte counts of 10,000 ± 4.500 cell/mm3 [The MIBO emphasised reporting cellular components from whole blood and deliverable process of PRP to understand influential characteristics of individual preparation . Even th3 , and 1,33 [3 . The oth3 [3 , 30 usedcell/mm3 .The number of studies of PRP treatment in OA ankle has been scarce. In this review, three before–after series supported that use of PRP was safe and effective , 13, 29,This sensitivity analysis did not find the relationship between traumatic OA or severe ankle OA and VAS. The explanation might be a limitation of previous surgical information, and reliability of Takakura and Kellgren classification . Most anNumerous classifications of ankle OA are not designed specifically to associate with the primary or secondary causes. Claessen et al.  reportedWhole blood platelets, differential leukocytes, and red blood cells should be analysed in all samples. Unfortunately, none of the included studies reported whole blood characteristics. Type and dose of anticoagulant is another concern. By chelating Ca+, ethylenediaminetetraacetic acid (EDTA) might disrupt function of platelets and might reduce the growth factors in the PRP prepared from EDTA-anticoagulated whole blood . Thus, tThree studies , 12, 29 Two included studies , 29 descAfter PRP injection into the tissue, the natural process of activation is commenced forming the fibrin network, and the plasma becomes the gel-like fibrin clot . Some inI2 93.01% and 58.93%, respectively. In the study by Repetto et al. [Total volume of injection has been recognised as one of influencing factors affecting the treatment outcome. Regarding to meta-regression of four included studies –13, 29, o et al.  administAge, immunity, metabolic disorders, and concomitant medication possibly impacted to cellular impairment, collagen deformation, microbiota changes, cellular viability and may contribute to therapeutic outcomes of PRP . From foFuture studies investigating the additive effects of collective doses are needed to support multiple injections. Intrinsic and extrinsic factors affecting PRP quality have to be explored. Moreover, standardised clinical and imaging outcomes should be assessed and compared between studies.Since this review was based on secondary data, the quality of evidence and data retrieval might be limited and affect the clinical application. Data were gathered as much as possible, and were all researchers agreed in searching, selection, and extraction by consensus. The quality of the studies, publication biases, and heterogeneity were assessed with standard statistical analysis to achieve the best available evidence. Limitations of this study are including descriptive case series with serious to critical risk of biases, publication biases or heterogeneity of the VAS outcome, very low-GRADE quality of evidences, inadequate data of quality of life for analysis and focused only on the maximum treatment effects at 12 weeks of follow-up. Included PRP studies in this review seemed to deliver a similar effect size as that of placebo from the previous RCT . RegardiTreatments of ankle OA with various PRP preparations seemed to be effective, in terms of pain and function, when compared with before treatment. With very low quality of evidences, high costs, and diverging settings, PRP is weakly recommended for ankle OA as an alternative or adjunct therapy after failed conservative treatment. Its benefits would be attained approximately 12 weeks after injection with acceptable minor complications.With regard to the hypothesis, the best available evidence of pooling different PRPs for ankle OA demonstrated only short-term, before–after pain, and functional improvement. Its benefit was similar to placebo effects of the included RCT. Further modified PRP preparation or limited use as alternative therapy may apply for ankle OA. The future studies investigating the outcomes of PRP injection for ankle OA should follow the MIBO consensus to make the processing of PRP preparation reproducible and to make the comparison of the outcomes between studies possible.Additional file 1: Table S1 Minimum Reporting Requirements for Clinical Studies Evaluating PRP Checklists for included studies. Table S2 Meta-regression of factors related to pain and functional score. Fig. S1 Meta-regression of association between platelet-rich plasma (PRP) volume and visual analog scale (VAS).  Fig. S2 Forest plots of visual analog scale at 12 weeks: A subgroup analysis by age groups. Fig. S3 A Funnel plot and B contour-enhanced funnel plot for visual analog scale. Fig. S4 Funnel plot for functional scores, SMD = standardised mean difference.


Preoperative anemia is a frequent complication in pancreatic surgical patients, and it adversely affects morbidity, mortality, and postoperative red blood cell (RBC) transfusion rates. Iron deficiency (ID) is often the underlying cause of anemia and constitutes a modifiable risk factor.Single-center, longitudinal prospective cohort study conducted between May 2019 and August 2022 at the University Medical Center Groningen in the Netherlands. Patients scheduled for pancreatic surgery were referred to the outpatient prehabilitation clinic for preoperative optimization of patient-related risk factors. Patients were screened for anemia (< 12.0 g/dL in women and < 13.0 g/dL in men) and ID . Intravenous iron supplementation (IVIS)  was administered to patients with ID at the discretion of the consulting internist. Pre- and postoperative hemoglobin (Hb) levels were assessed, and perioperative outcomes were compared between patients receiving IVIS (IVIS-group) or standard care (SC-group).p < 0.001, and 11.8 versus 13.4, p < 0.001, respectively) did not exist at discharge . Preoperative IVIS led to a significant increase in mean Hb levels . Fewer SSI were observed in the IVIS-group , which remained significant in multivariable regression analysis (OR 7.01 (1.68 – 49.75), p = 0.02).From 164 screened patients, preoperative anemia was observed in 55 (33.5%) patients, and in 23 (41.8%) of these patients, ID was the underlying cause. In 21 patients, ID was present without concomitant anemia. Preoperative IVIS was administered to 25 patients, out of 44 patients with ID. Initial differences in mean Hb levels (g/dL) between the IVIS-group and SC-group at the outpatient clinic and one day prior to surgery (10.8 versus 13.2, ID is prevalent in patients scheduled for pancreatic surgery and is amendable to preoperative correction. Preoperative IVIS increased Hb levels effectively and reduced postoperative SSI. Screening and correction of ID is an important element of preoperative care and should be a standard item in daily prehabilitation practice.The online version contains supplementary material available at 10.1186/s13741-023-00323-1. Iron deficiency (ID) is the most common cause of anemia in surgical cancer patients and amendable to preoperative correction  were excluded from the final analysis.As part of the Frail-study, all patients scheduled to undergo hepatopancreatobiliary surgery are referred to the prehabilitation outpatient clinic to screen for patient related modifiable risk-factors. The prehabilitation outpatient clinic visit is integrated in the preoperative work-up of patients. The method of screening and assessment at the prehabilitation outpatient clinic has been published previously , and C-reactive protein (CRP) were standard items in the preoperative laboratory screening. The Hb level was measured using a Sysmex XN10 analyzer . Furthermore, serum iron was measured using a colorimetric assay, ferritin was measured using immunoassay, and transferrin was measured using an immunoturbidimetric assay . TSAT was calculated as 100 × serum iron (μmol/L) / (25.2 × transferrin (g/L)). Anemia was defined as an Hb level of < 12.0 g/dL in women and < 13.0 g/dL in men WHO . ID was Patient characteristics, laboratory results, time between IVIS and surgery, and clinical outcome data were collected from patients’ electronic medical records. The primary endpoint of this study was the change in Hb levels over time. For this, blood was collected from patients during the first prehabilitation outpatient clinic visit (T0), at admission (T1), and at discharge (T2). Iron parameters  were solely evaluated during the outpatient clinic visit. The secondary endpoints included the need for postoperative RBC transfusion, surgery-specific postoperative complications up to 30 days after surgery, length of hospital stay, and unplanned readmission rate. RBC transfusion was defined as any allogenic RBC transfusion during the postoperative hospital stay. Postoperative pancreatic fistula (POPF), post pancreatectomy hemorrhage (PPH), delayed gastric emptying (DGE), bile leakage (BL), and postoperative chyle leakage (CL) were defined and graded according to ISGPS and ISGLS classifications . All models yielded an estimated regression coefficient (β), with a corresponding 95% confidence interval for the hazard ratio and odds ratio with 95% confidence interval. The R software package, version 4.2.2.  was used for statistical analysis, using the ggpubr, tidyverse, and ggplot2 packages. In all analyses, a p-value < 0.05 was considered statistically significant.The normality of continuous data was checked using the Shapiro–Wilk test and QQ-plots. Continuous variables are presented as mean with standard deviation (SD) or as median with interquartile range (IQR) based on normality of distribution. Categorical data are presented as numbers and percentages. Differences between groups were calculated using the student’s t-test, Mann–Whitney U test, Chi-squared test, or Fisher exact test, as appropriate. Furthermore, mean differences in specific follow-up points of Hb levels between groups were determined using one-way ANOVA and Tukey’s range test. Multivariable logistic regression analysis with stepwise backward elimination was performed to assess the effect of iron supplementation on the occurrence of SSI. The eligible variables for the adjusted model were selected when the univariable analysis yielded a A total of 164 patients were analyzed for ID and IDA, of whom 25 were referred for IVIS , predominantly female , and had a mean BMI of 27.5 kg/m2 . The percentage of patients with an ASA classification 3 was higher in the IVIS-group . In contrast, the incidence of tobacco abuse was higher in the SC-group . Other relevant patient characteristics were well balanced between groups.The patient characteristics for both study groups are presented in Table p < 0.001) and TSAT 12.9% versus 25.9% (p < 0.001) in the IVIS- and SC-group, respectively (Table p < 0.001). This difference in mean Hb level remained statistically significant at admission  yet no longer existed at discharge  (Table p = 0.03). It is worth noting that in both groups, a significant decrease in mean Hb level occurred between admission and discharge .As expected, preoperative iron parameters were significantly lower in the IVIS-group, with a ferritin level of 88 μg/L versus 216 μg/L (p = 0.07). Also, the rate of RBC transfusions administered in the IVIS-group was slightly higher . The median length of hospital stay was 12 days versus 11 days in the IVIS-group and SC-group, respectively, p = 0.23. The rate of surgery-specific complications and cardiopulmonary complications did not differ between groups. In the SC-group, significantly more SSI  were observed . This effect remained significant when tested  for the higher tobacco abuse incidence in the SC-group in a multivariable logistic regression analysis (Table The types of pancreatic resections and rate of complementary (vascular) resection was similar between groups Table . Albeit p = 0.04) (Table Sp = 0.003). When comparing the laboratory results (Table Sp < 0.001) and 13.0 g/dL at admission . However, while the preoperative Hb level significantly increased after IVIS in the IVIS-group, in the nontreated ID-group, a decline in Hb level was observed . At discharge, Hb levels between groups were comparable . In the nontreated ID-group, the incidence of POPF  and SSI  was significantly higher.Results of a subgroup analysis comparing the IVIS-group to the nontreated ID-group is available in the supplementals. The mean age in this subgroup analysis was higher in the IVIS-group  Table S. MoreoveIn this study, we demonstrated an important role of preoperative IVIS in pancreatic surgery patients with IDA and ID. Preoperative anemia was present in more than 30% of the patients, and in more than 40% of these patients, ID was the underlying cause of anemia. In around 20% of the nonanemic patients, ID was present, making these patients also susceptible for anemia. In 25 patients, preoperative IVIS led to a significant increase in mean preoperative Hb level (from 10.8 g/dL to 11.8 g/dL), which resulted in an almost equal mean Hb level at discharge compared to patients who did not receive preoperative IVIS , suggesting high effectiveness of preoperative IVIS. This preoperative lift in mean Hb levels might have enhanced postoperative outcomes in these patients, resulting in similar surgery-specific complication rates compared to patients without IDA. Moreover, a clinically relevant reduction in SSI was observed in the IVIS-group. No differences in postoperative RBC transfusions were observed. However, postoperative RBC transfusion demand is greatly influenced by the amount of intraoperative blood loss, and the higher median intraoperative blood loss in the IVIS-group will have had a clinically relevant impact on transfusion demands.Our results are supported by previous studies demonstrating the beneficial effect of preoperative IVIS on increasing preoperative Hb levels in various surgical populations   and reduced cardiac output reserve during exercise, resulting in diminished exercise capacity , this effect remained significant. Moreover, iron supplementation might have had a beneficial effect. Wound tissue macrophages play an essential role in effective wound healing by orchestrating tissue repair  could not be investigated. Finally, the effect of IVIS on restoring iron deposits was not evaluated, as iron parameters were only assessed at the outpatient clinic. Moreover, this study lacked other relevant iron parameters  which can adequately assess ID and restoration of iron deposits in red blood cells in inflammatory conditions. Nevertheless, a clinically adequate increase in mean Hb levels was observed in this study. For future studies, ideally a randomized controlled trial with an adequate sample size comparing the effect of IVIS versus standard care in two ID patient cohorts would be the preferred study design.In conclusion, the results of this study demonstrate a high incidence of ID in pancreatic surgical patients and the potential of preoperative IVIS to increase mean Hb level. ID constitutes a significant risk factor in patients, which is amendable to preoperative correction. In addition to increasing mean Hb levels, IVIS led to a reduction in postoperative SSI. However, as we observed in this study, protocol adherence to refer patients for preoperative IVIS is suboptimal. We have established that ID is a serious issue in pancreatic surgical patients, and therefore we conclude that preoperative screening and correction of ID are important elements of preoperative care and should be standard items in daily prehabilitation practice.Additional file 1: Table S1. Characteristics of patients receiving IVIS and patients with nontreated ID. Table S2. Detailed overview of anemia and iron parameters in patients receiving IVIS and patients with nontreated ID. Table S3. Surgical details and postoperative outcomes.


First, to assess the number of patients with anorectal malformations (ARM) in whom additional urological and/or gynecological anomalies were identified through routine screening with cysto- or vaginoscopy prior to reconstructive surgery. Second, to assess potential procedure-related complications.Retrospective mono-center cohort study, including all ARM patients born between January 2019 and December 2022. Routine screening consisted of cystoscopy for male patients, with the addition of vaginoscopy for female patients. Chi-square was used to compare the screening percentages over time.In total, 38 patients were included, of whom 27 (71.1%) underwent cystoscopy ± vaginoscopy, without the occurrence of complications. Nine of 13 females (69.2%) underwent cysto- and vaginoscopy and 18 of 25 males (72.0%) underwent a cystoscopy. The percentage of patients that underwent these procedures improved over the 2 time periods . In 15 of 27 patients (55.6%) that underwent cystoscopy ± vaginoscopy, additional anomalies were found that were not identified through physical examination or US-kidney.In 56% of the patients that underwent cysto- ± vaginoscopy, additional anomalies were identified that were not with imaging studies or physical examination. This study emphasizes the potential benefit of routine cysto- and vaginoscopy in the diagnostic work-up of children with ARM.Level of Evidence: III.The online version contains supplementary material available at 10.1007/s00383-023-05565-0. Anorectal malformations (ARM) are rare congenital anomalies occurring in approximately 1–3 per 5000 live born children . ARM canAdditional urogenital anomalies are identified in approximately 50% of ARM patients, and can be divided into urological anomalies  and gynecological anomalies  . FurtherAwareness of the potentially associated urogenital anomalies has increased over time, but discussion remains about the optimal diagnostic screening strategy and timing  . ComprehA retrospective cohort study from a database maintained since late 2018 (using Castor EDC (Electronic Data Capture) software ) was perThis study was reviewed by the medical ethical commission and was not subject to the WMO statement (ref. no. W20_230 #20.576). Regarding the primary Emma Children’s Hospital database, written information was provided to parents or legal guardians for all identified ARM patients, including a letter of objection. In case of objection, patients were removed from the database (n = 6).Data on patient characteristics, type of ARM, reconstructive surgery , posterior sagittal anorectoplasty (PSARP), other reconstructive interventions), additional anomalies , preoperative screening , peroperative findings, and complications associated with cysto- and vaginoscopy were extracted from the database by two independent researchers . In case of disagreement between researchers, a third researcher (CK and/or RG) was consulted for final decision.Since 2018, routine screening to identify urogenital anomalies in Amsterdam UMC consisted of thorough physical examination to assess genital anomalies, ultrasound of the urogenital tract within 4 weeks of birth, cystoscopy , and vaginoscopy  prior to the intended reconstructive surgery, proceeding immediately to reconstruction . Additionally, VCUG and MRI were performed on indication only. Indications for VCUG are dilated ureters and/or kidneys on US, febrile UTI, and fistula identification, and for MRI undetermined US findings or in doubt on anatomy , that underwent routine screening with cystoscopy ± vaginoscopy, and in whom new additional urological and/or gynecological anomalies were identified prior to reconstructive surgery.Secondary outcome was the number of ARM patients in whom complications occurred during or after routine cystoscopy and/or vaginoscopy.Statistical analysis was conducted using IBM SPSS Statistics for Windows, Version 28 . Descriptive statistics were used for analysis of baseline characteristics. These were reported as proportions and percentages for binary or categorical variables, and as mean with standard deviation (SD) or as median with interquartile range (IQR) for continuous variables as appropriate. To evaluate changes over time in screening, patients were categorized into 2 time periods . To compare screening percentages over time, because of little numbers, Fisher’s exact was used. A p value of < 0.05 was considered statistically significant. Missing or unknown data were described. No quantitative syntheses were performed due to small numbers.n = 3), Caudal regression syndrome (n = 3), and Townes − Brocks syndrome (n = 2) were most common. Spinal cord anomalies were identified in 9 of 38 patients (23.7%), with tethered spinal cord most often identified in 4 of 9 patients (44.4%), of whom, due to age, only 1 patient underwent urodynamic research to rule out neurogenic bladder disorder. Through physical examination, additional anomalies were identified in 2 of 13 female patients (15.4%), encompassing the presence of a vaginal tag (n = 1), and an enlarged clitoris and fused labia (n = 1), and additional anomalies were identified in 6 of 18 male patients (33.3%), encompassing hypospadia (n = 3), undescended testes (n = 3), and a bifid scrotum (n = 1). ASARP was the most performed reconstructive technique in 23 of 38 patients (60.5%), with median age of 3.5 months (IQR 3.0–5.25) at date of surgery. No patients underwent reconstructive surgery within the first week of life. One patient (2.6%) died during the study period due to bacterial meningitis, at 6 months of age. An overview on patient characteristics can be found in Table In total, 43 patients were identified in the Castor EDC database of whom 5 were excluded because no reconstructive ARM surgery was yet performed. Consequently, 38 patients were included for analysis. The cohort comprised 13 female (34.2%) and 25 male patients (65.8%) with a median age of 15.5 months (IQR 9.0–30.0) at latest follow-up. Seven different ARM types were identified, of which recto-perineal fistula occurred most often, encompassing 8 of 13 female patients (61.5%) and 13 of 25 male patients (52.0%). VACTERL association or syndromic anomalies were identified in 11 of 38 patients (28.9%), of which VACTERL-association (n = 3), bicorporeal uterus (n = 2), and ectopic kidney (n = 2) occurring most often. VCUG was performed in 8 of 38 patients (21.1%) to localize the fistula (n = 5), and because of dilated kidneys on US (n = 3), resulting in identification of VUR  in 1 of 8 patients (12.5%). A specification of patients with additional anomalies identified through US-kidney and VCUG can be found in Supplementary Material 1.Almost all ARM patients (35 of 38 patients (92.1%)) underwent US-kidney prior to reconstructive surgery, resulting in identification of additional anatomical urogenital anomalies in 12 of 35 patients (34.2%), with hydronephrosis . In 11 patients with recto-perineal fistula (n = 8), anal stenosis (n = 2), or imperforate anus (n = 1), cysto- and/or vaginoscopy were not performed without reasons available in the patient’s medical chart. In total, 23 of 35 patients (65.7%) had a urogenital anomaly (identified through either imaging studies or cysto- and/or vaginoscopy). In addition, long-term follow-up by a pediatric urologist was performed in all patients in whom any anomaly was identified through cystoscopy.In total, 27 of 38 patients (71.7%)  underwent routine screening with cysto- and/or vaginoscopy prior to reconstructive surgery, resulting in identification of urogenital anomalies in 15 patients (55.6%). No procedure related complications during or after cystoscopy or vaginoscopy were encountered, including no postoperative UTI’s. Over the two time periods, the percentage of patients that underwent full screening increased (n = 2) and a short urethra (1.2 cm) (n = 1). Additionally, in 1 patient (2 day old with an H-type fistula), the urethral orifice could not be identified through cystoscopy, requiring a combined cysto-vaginoscopy through the H-fistula for definitive identification of the urethral orifice. Through vaginoscopy, additional anomalies were identified in 2 of 11 patients (18.2%), encompassing vaginal atresia (n = 1) and a short longitudinal septum between cervices (n = 1). The vaginoscopy finding of vaginal atresia caused the planned reconstructive surgery (ASARP) to be postponed. An overview of female patients with additional anomalies identified in the urogenital tract through cysto- and/or vaginoscopy prior to reconstructive surgery, according to type of ARM is shown in Table In total, 9 of 13 female patients 69.2%) underwent routine screening including cysto- and vaginoscopy prior to reconstructive surgery, resulting in identification of additional urogenital anomalies in 4 of 9 patients (44.4%). Two of 13 patients (15.4%) underwent only vaginoscopy, and 2 of 13 patients (15.4%) underwent no screening by endoscopy prior to reconstructive surgery. Through cystoscopy, additional anomalies were identified in 4 of 9 patients (44.4%), encompassing bladder wall trabeculation (% underwen = 7), and bladder wall trabeculation (n = 3) occurring most often. All patients with PUV or syringocele underwent surgical incision by the pediatric urologist to resolve the urethral obstruction during cystoscopy, without the occurrence of complications. No planned reconstructive surgeries were postponed due to cystoscopy findings. An overview of male patients with additional anomalies in the urogenital tract identified through cystoscopy prior to reconstructive surgery according to type of ARM is shown in Table In total, 18 of 25 male patients (72.0%) underwent routine screening including cystoscopy prior to reconstructive surgery, identifying additional anomalies in 11 of 18 patients (61.1%), with urethral obstruction (syringocele or PUV)  underwent routine cysto- and/or vaginoscopy prior to reconstructive surgery, resulting in identification of additional urogenital anomalies in 44% and 61%, in the female and male population, respectively. Additional urogenital anomalies were identified in both ARM patients with both simple and more complex types. No complications during or after cysto- or vaginoscopy were encountered. In 56% of the patients that underwent cysto- and/or vaginoscopy additional anomalies were identified that were not suggested in history, seen with physical examination, or encountered on earlier imaging studies.In this study, approximately 65% of ARM patients had a urogenital anomaly. Accurate data on the prevalence of urogenital anomalies in ARM patients is limited, but previous large cohort studies have described a prevalence of approximately 50% , 19–21. Despite implementation of routine cystoscopy ± vaginoscopy as routine screening in ARM patients since late 2018 in our center, these procedures were not performed in 7 male (28%) and 4 female (31%) patients, respectively. However, over time, the number of patients that underwent these procedures has increased greatly (to 90%). This could be explained by the fact that implementation of novel (diagnostic) strategies into clinical care pathways take some time as it first has to overcome so called obstructing factors . In addiEmbedding routine cysto- and/or vaginoscopy in standard care might be beneficial for early identification of additional urogenital anomalies in patients with ARM, both for (specific) treatment and follow-up, as well as for patient and parental counseling when anomalies are found. In this study, none of the patients suffered any hazard from the cystoscopy of vaginoscopy. In the previous studies investigating cysto- and vaginoscopy in different patients than ARM, similar results of low complication rates were reported for cysto- and vaginoscopy –26. FurtThis study should be interpreted in light of some strengths and limitations. First, to our knowledge this is the first study evaluating outcomes of standard cysto- and/or vaginoscopy prior to reconstructive surgery in ARM patients. Second, we were able to show that in half of the patients that underwent cysto- or vaginoscopy, new urogenital anomalies were identified, which were not identified through physical examination and history or with imaging studies. By incorporating routine cysto- and/or vaginoscopy prior to reconstructive surgery, this might lead to earlier diagnosis and thus earlier treatment . However, although cysto- and vaginoscopy were performed by a specialized multidisciplinary team with a key role for the pediatric urologist, the identified urogenital anomalies might be subject to inter observer variability. Furthermore, due to the retrospective nature of this study, information and selection bias are likely present. To reduce this to a minimum, data was extracted by the primary researcher (CB) with a data check by another coauthor (TM) and all patients were consecutively included between 2019 and 2022. Because ARM is extremely rare, only a small number of patients could be included and extensive statistical analyses were not possible. Even though numbers are small, it is important to report these data, to open a discussion on whether or not routine cysto- and/or vaginoscopy is indicated and of importance as standard care in patients with ARM.In conclusion, 71% of our ARM patients underwent routine cysto- and vaginoscopy in female patients and cystoscopy in male patients prior to reconstructive surgery, without occurrence of complications. In more than half of the patients that underwent these procedures, new urogenital anomalies were identified through cysto- or vaginoscopy that were not found with history, physical examination or imaging studies. This emphasizes the potential benefit and importance of routine cystoscopy and/or vaginoscopy in the diagnostic work-up prior to reconstructive surgery in patients with ARM.Supplementary file1 (DOCX 16 KB)Below is the link to the electronic supplementary material.