BACKGROUND:The vaginal microbiota test predicts the success of in vitro fertilization (IVF), but with no therapy available to improve a low profile, couples must decide whether to proceed or postpone treatment. We aim to examine how couples interpret vaginal microbiome results and make postponement decisions within a shared decision making (SDM) framework. METHODS:Women undergoing IVF or IVF-intracytoplasmic sperm injection (IVF-ICSI) treatment at 2 Dutch hospitals received the ReceptIVFity test™, which classified the vaginal microbiome as high (52.6% chance of conception), medium (23.6%), or low (5.9%) profile based on predicted implantation success after a fresh embryo transfer. Physicians discussed the results with couples using SDM, after which the couples decided whether to proceed or postpone treatment. The primary outcome was the patients' perceived involvement in shared decision making, assessed with the SDM-Q-9 questionnaire. The secondary outcome was the proportion of couples postponing treatment after a low microbiome profile. RESULTS:Between October 2018 and November 2020, 728 women were enrolled. SDM-Q-9 responses showed high perceived involvement overall but lower scores for "exploring options," reflecting limited alternatives when the choice is to proceed or postpone treatment. A low profile was found in 35.4% (258/728). After the SDM consultation, 49.6% (128/258) chose to postpone treatment, with postponement rates increasing to over 80% among couples in later IVF cycles. Decisions were influenced by personal, emotional, and practical considerations, including the Dutch insurance reimbursement system (3 insured IVF or IVF-ICSI cycles regardless of postponement) and the absence of effective treatment to modify a low profile. CONCLUSIONS:These findings demonstrate that couples can understand and use prognostic information when supported by SDM and that the ReceptIVFity test™ facilitated discussion about chances of success, timing of treatment, decisions to proceed or postpone, and personal values.

Introduction:Prematurity is associated with intestinal immaturity and gut microbiota alterations, both of which are linked to necrotizing enterocolitis (NEC) and sepsis. An important yet understudied contributor in the development of the gastrointestinal (GI) tract is amniotic fluid (AF). The aim of this study is to assess the composition of AF collected during extremely preterm birth. Secondary objectives are to identify AF profiles of pregnancies complicated with chorioamnionitis and/or fetal growth restriction, assess key AF components across gestation, correlate AF profiles with neonatal outcomes (e.g., NEC and sepsis), and explore associations with neonatal gut microbiota. Methods and analysis:In this multicenter prospective cohort study, AF (∼5 mL) will be collected from obstetric patients delivering their infants extremely preterm [gestational age (GA) 24 + 0/7-27 + 6/7 weeks, n = 125]. AF can be collected safely and non-invasively during vaginal delivery or cesarean section. AF samples will also be collected from a reference group (GA <23 + 6/7 and 28 + 0/7-40 + 6/7 weeks, n = 150). Characterization of AF will include microbial and metabolic profiling. By advancing our understanding of the role of AF in the GI development and neonatal disease, this study may contribute to the early identification of infants at risk to develop NEC and/or sepsis. Ultimately these findings may facilitate early targeted microbiota-based interventions to prevent disease progression and improve outcomes. While the current study provides clinical opportunities, several methodological challenges inherent to collecting AF at birth must be acknowledged, including variability in sampling method, maternal blood interference, and the risk of microbial contamination. Ethics and disseminations:Ethical approval was received by the METC of the Máxima Medical Center in Veldhoven, the Netherlands (W24.042). The study was registered in a public clinical trial registry (NCT07152106). Study findings will be disseminated through peer-reviewed journal articles as well as national and international conference presentations. Clinical Trial Registration:https://clinicaltrials.gov/study/NCT07152106, identifier NCT07152106.

The diagnosis of pleural infection has traditionally relied on microbiological culture, a method often limited by slow bacterial growth and difficulties in culturing fastidious organisms. This study introduces Molecular Culture ID, a rapid and comprehensive molecular technique capable of detecting and identifying a wide range of bacteria down to the species level. By analyzing 440 pleural effusion samples derived from patients suspected of pleural infection and comparing the outcome to routine diagnostics, we demonstrate that Molecular Culture ID significantly outperforms traditional culture. Molecular Culture ID identified 133 positive samples, compared to 55 positive samples identified by traditional culture. The 78 additional positive samples detected by Molecular Culture contained important pathogens including species such as Streptococcus pneumoniae, Escherichia coli, and Staphylococcus aureus. These findings highlight Molecular Culture ID as a promising alternative for the rapid and accurate diagnosis of pleural infections, offering a substantial improvement over conventional methods.

Gut microbiota changes are associated with inflammatory bowel disease (IBD). In most microbiota studies, intestinal microbiota composition is examined in fecal samples, but their representativeness of mucosa-associated microbiota at inflammation sites remains unclear. This study aimed to explore microbiota composition in different IBD sample types and assess their interchangeability for research and clinical use. Multicentre, prospective, observational study including 200 IBD patients (518 samples). We compared microbiota profiles from faeces, rectal swabs and mucosal biopsies from different colon sites. Microbiota composition was analyzed by Molecular Culture™, a bacterial profiling technique based on species-specific differences in the 16S-23S interspace region of bacterial ribosomal DNA, with taxonomic classification by phylum-specific fluorescent PCR primers. Fecal samples and rectal swabs contained a higher microbial diversity and abundance than colonic biopsies. Microbiota compositions of different sample types within an individual happened to be quite dissimilar (median cosine similarities 0.43-0.53). Within individuals, biopsies from the same location in the colon were just as similar as biopsies from different locations (median cosine similarities 0.85 vs. 0.82, respectively). Fecal samples, rectal swabs and colonic biopsies of an individual show distinct microbiota profiles and should not be used interchangeably in microbiota studies or clinical applications.

Bacterial peritonitis (BP) is a serious complication commonly associated with cirrhosis and ascites, often leading to high mortality rates. Although these effects could be reduced with timely and appropriate antibiotics, traditional BP diagnosis relies on culture, often delaying targeted treatment. Therefore, the use of fast molecular assays holds the potential to enhance laboratory diagnosis. In this study, we assessed the diagnostic performaance of Molecular Culture ID, a broad PCR-based assay targeting the 16S-23S interspace rDNA region in the scope of BP diagnosis. The residual material from 247 peritoneal fluid samples submitted for routine diagnostics was analyzed using Molecular Culture ID and compared alongside the standard of care (SOC) results. Sample positivity and species identification outcomes of Molecular Culture ID were compared to those of SOC. Molecular Culture ID yielded 1.6x more positive samples than SOC. Percent positive agreement (PPA) between Molecular Culture ID and SOC at the sample level was 90.1% (IC 95%, 81.0% to 95.1%), and negative percent agreement (NPA) was 70.5% (IC 95%, 63.3% to 76.7%). At the species level, the PPA was 75.2% (95% CI 67.2% to 81.8%). Molecular Culture ID yielded 289 extra bacterial identifications, mainly anaerobic species. High leukocyte counts, indicative of infection, were concordant with Molecular Culture ID positivity. Molecular Culture ID demonstrated enhanced BP diagnostic capabilities compared to SOC, with higher positivity rates, more comprehensive species identification for difficult to culture species and a high correlation with leukocyte counts.

[This corrects the article DOI: 10.3389/fcimb.2025.1645965.].

Background and Aims Fecal calprotectin (FCP) has limited specificity as diagnostic biomarker of pediatric inflammatory bowel disease (IBD), leading to unnecessary invasive endoscopies. This study aimed to develop and validate a fecal microbiota and amino acid (AA)-based diagnostic model.Methods Fecal samples from a discovery cohort (de novo IBD and healthy controls [HC]) were used to develop the diagnostic model. This model was applied in a validation cohort (de novo IBD and controls with gastrointestinal symptoms [CGI]). Microbiota and AAs were analyzed using interspace profiling and liquid chromatography-mass spectrometry techniques, respectively. Machine learning techniques were used to build the diagnostic model.Results In the discovery cohort (58 IBD, 59 hC), two microbial species (Escherichia coli and Alistipes finegoldii) and four AAs (leucine, ornithine, taurine, and alpha-aminoadipic acid [AAD]) combined allowed for discrimination between both subgroups (AUC 0.94, 95% CI [0.89, 0.98]). In the validation cohort (43 IBD, 38 CGI), this panel of six markers could differentiate patients with IBD from CGI with an AUC of 0.84, 95% CI [0.67, 0.95]). Leucine showed the best diagnostic performance (AUC 0.89, 95% CI [0.81, 0.95]).Conclusions Leucine might serve as adjuvant noninvasive biomarker in the diagnostic work-up of pediatric IBD. Future research should investigate whether the combination of leucine with FCP could improve specificity and may help tailor the course of diagnostics.

Introduction: The low incidence of intradialytic hypotension (IDH) in high-volume (HV) hemodiafiltration (HDF) may help in maintaining gut perfusion during treatment. Preservation of gut endothelial integrity would limit or prevent bacterial translocation and subsequent systemic inflammation, which may contribute to the low mortality rate in HV-HDF. Methods: Forty patients were cross-over randomized to standard (hemodialysis [HD]) (S-HD), cool HD (C HD), and HDF (low-volume [LV] and HV, convection volume (CV) of 15 L and >= 23 L per session, respectively), each for 2 weeks. Quantitative assessment of microbial DNA (mDNA) in blood was performed before and after dialysis by 16S to 23S interspace profiling after DNA isolation. The intradialytic acute phase response (APR) was evaluated by high-sensitivity C-reactive protein (hs-CRP), interleukin-6 receptor (IL-6R), soluble CD14 (sCD14), and vascular-cell-adhesion molecule-1 (VCAM-1). Differences between modalities were primary objectives. Results: mDNA was absent from all samples. IL-6R, sCD14, and VCAM-1 increased equally in all modalities (median increase: 12.5%, 14.0%, 14.8%, respectively; P < 0.05). hs-CRP increased only in C-HD and HV-HDF (median increase: 12.6%, P < 0.05). After correction for hemoconcentration, most APR markers decreased (median: sCD14,-11.3% and VCAM-1,-14.4% in all modalities; IL-6R,-13.4% in C-HD, LV-HDF, and HVHDF; P < 0.05). hs-CRP only decreased in C-HD (-13.5%, P = 0.004). Conclusion: From this study, we conclude as follows: (i) circulating mDNA could not be demonstrated; (ii) in the crude analysis, a similar APR was noted in all modalities, individual markers remained stable or declined after correction for hemoconcentration; and (iii) because neither bacterial translocation nor an APR was observed in either modality, it is highly unlikely that the superior survival of HV-HDF is explained by a superior preservation of gut integrity.

IntroductionCladribine tablets are an effective treatment for relapsing remitting multiple sclerosis (RRMS). However, almost half of the treated patients are not free of disease activity after two years. The aim of this study was to describe the changes that cladribine tablets effectuate in the gut and oral microbiota and the peripheral immunological profile between responders and non-responders.MethodsIn this pilot study of the multicenter, prospective, observational BIA (Brain-Immune-Intestine Axis) study, we included patients aged 18 to 55 years with RRMS who were scheduled to start treatment with cladribine tablets. We assessed the clinical status and the immunological and microbiological profile prior to the start of the treatment and after three and twelve months. At twelve months, we assessed the response status, based on clinical relapses, radiological activity and disability progression on the Expanded Disability Status Scale.ResultsThe first twenty-five patients of the BIA study were included in this analysis. Ten patients (40%) were responders twelve months after treatment. Three months after treatment we found a significant decline of naïve and transitional B cells and memory B cells, and of CD57+ CD56dim NK cells. After twelve months the values recovered to baseline levels, except for the memory B cells. We did not find significant changes of the microbiological profile over time, except for a decline of the phylum Bacteroidetes in the oral samples twelve months after treatment. Baseline values and changes over time did not significantly differ between responders and non-responders. However, several phyla, genera or species (Bacteroidetes, Prevotella, Faecalibacterium prausnitzii) showed a higher relative abundance, and several phyla, genera or species (Proteobacteria, Escherichia coli) had a lower relative abundance in responders compared to non-responders.DiscussionAfter treatment with cladribine tablets, we found significant changes in the immunological landscape. Also, the microbiological profile showed several differences in microbes with known anti- or pro-inflammatory properties between responders and non-responders. Overall, we showed that we can measure a treatment effect from cladribine tablets with our analyses. Future research on data from the BIA study, with a larger sample size and extended follow-up, can possibly confirm the reliability of our findings.

Since its first appearance, severe acute respiratory syndrome coronavirus 2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein. A highly selective multiple reaction monitoring-cubed (MRM3) strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral nucleocapsid protein peptides from nasopharyngeal swabs samples presenting a cycle threshold value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immunopurification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with cycle threshold values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted MS.

Clostridioides difficile infection (CDI) is the most common cause of antibiotic-associated diarrhoea. Fidaxomicin and fecal microbiota transplantation (FMT) are effective, but expensive therapies to treat recurrent CDI (reCDI). Our objective was to develop a prediction model for reCDI based on the gut microbiota composition and clinical characteristics, to identify patients who could benefit from early treatment with fidaxomicin or FMT. Multicentre, prospective, observational study in adult patients diagnosed with a primary episode of CDI. Fecal samples and clinical data were collected prior to, and after 5 days of CDI treatment. Follow-up duration was 8 weeks. Microbiota composition was analysed by IS-pro, a bacterial profiling technique based on phylum- and species-specific differences in the 16–23 S interspace regions of ribosomal DNA. Bayesian additive regression trees (BART) and adaptive group-regularized logistic ridge regression (AGRR) were used to construct prediction models for reCDI. 209 patients were included, of which 25

IntroductionAppendicitis is one of the most common causes of acute abdominal surgery in children. The clinical course of appendicitis ranges from simple to complex appendicitis. The mechanisms underlying the heterogeneity of appendicitis in children remain largely unclear. Dysregulated T cell responses play an important role in several inflammatory diseases of the intestine, but the extend of T cell dysregulation in appendicitis in children is less well known.MethodsTo characterize appendiceal T cells in simple and complex appendicitis we performed in-depth immunophenotyping of appendiceal-derived T cells by flow cytometry and correlated this to appendiceal-derived microbiota analyses of the same patient.ResultsAppendix samples of twenty children with appendicitis (n = 8 simple, n = 12 complex) were collected. T cells in complex appendicitis displayed an increased differentiated phenotype compared to simple appendicitis, including a loss of both CD27 and CD28 by CD4+ T cells and to a lesser extent by CD8+ T cells. Frequencies of phenotypic tissue-resident memory CD69+CD4+ T cells and CD69+CD8+ T cells were decreased in children with complex compared to simple appendicitis, indicating disruption of local tissue-resident immune responses. In line with the increased differentiated phenotype, cytokine production of in particular IL-17A by CD4+ T cells was increased in children with complex compared to simple appendicitis. Furthermore, frequencies of IL-17A+ CD4+ T cells correlated with a dysregulation of the appendiceal microbiota in children with complex appendicitis.ConclusionIn conclusion, disruption of local T cell responses, and enhanced pro-inflammatory Th17 responses correlating to changes in the appendiceal microbiota were observed in children with complex compared to simple appendicitis. Further studies are needed to decipher the role of a dysregulated network of microbiota and Th17 cells in the development of complex appendicitis in children.

The composition of the vaginal microbiota prior to an IVF/IVF-ICSI treatment can predict the chance of achieving a pregnancy. To improve clinical applicability and be more patient-friendly, the self-collection of vaginal samples would be preferable. However, the reliability of patient-collected samples compared to physician-collected samples remains unclear. This study compares microbiome outcomes from patient-collected versus physician-collected vaginal samples. This is a prospective pilot study consisting of two cohorts: Cohort I involved patient self-sampling of the vagina, followed by a physician-collected vaginal swab, while Cohort II involved the reversed order of collection. The interspace profiling (IS-Pro) technique was used to analyze the microbiota composition in all samples. From May 2021 to March 2022, a total of 444 samples were collected from n = 222 patients (aged 21–44 years), with Cohort I (n = 109) and Cohort II (n = 113). The vaginal microbiome composition of both cohorts was highly similar, regardless of the sampling order, with a mean cosine similarity of 0.93 (95% CI 0.91, 0.95) in Cohort I and 0.94 (95% CI 0.92, 0.96) in Cohort II. Furthermore, ANOVA analysis revealed no significant differences in bacterial species abundance between physician- and patient-collected samples, nor between first and second sample collections. The self-collection of vaginal samples can be considered comparable to physician-collected samples and indicates a more patient-friendly and convenient collection of the vaginal microbiome in an outpatient clinical setting.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles.

Infectious complications and anastomotic leakage after colorectal resection are common1–3, impede postoperative recovery, and influence oncological outcome4. The patient’s gut microbiota has a critical role in metabolic function, protection against pathogens, and modulation of immune function5. In addition, the microbiota plays a pivotal role in the development of infectious complications after gastrointestinal surgery6,7. One phylum which normally represents less than 10 per cent of the bacteria in the gut is Proteobacteria8. During the perioperative phase, the gut microbiota composition is disturbed by various factors, including surgery itself, mechanical bowel preparation, and antibiotics1,9–11. These disturbances cause dysbiosis, which can lead to overgrowth of normally low-abundant pathogenic species, such as Proteobacteria12. Clinical studies in humans elucidating the role of this phylum in postoperative infectious complications remain scarce. The randomized SELECT trial showed a reduction in postoperative infectious complications after elective colorectal surgery with selective decontamination of the digestive tract (SDD) as oral antibiotic prophylaxis1. SDD was administered to reduce the pathogenic bacterial load, including Proteobacteria, in the gut. This study, a subgroup analysis of patients who participated in the SELECT trial, aimed to determine the role of preoperative proteobacterial load in the gut in postoperative infectious complications.

BACKGROUND:The treatment algorithm in late-onset inflammatory adverse events with soft-tissue fillers depends primarily on the assumed causative factor: immunologic or bacterial.METHODS:The authors included 29 patients, 13 of whom experienced late-onset inflammatory adverse events to fillers (inflammatory group) and 16 who did not (reference group). Biopsies were acquired from both groups with an 18-G needle. Before taking the biopsy, the authors acquired skin swabs for 25 of the 29 patients. The IS-pro method-a new and very sensitive method to detect microbiota-was used. This is a novel broad-range polymerase chain reaction technique based on length and sequence variations of the 16S to 23S ribosomal interspacer region. IS-pro can detect bacteria at low abundances and identify them up to species level. To exclude contamination from skin microbiota, the authors compared the microbiota found on skin swabs with that found in the corresponding biopsies.RESULTS:A high level of Gram-positive bacteria was found in biopsies of soft-tissue fillers, predominantly in patients from the inflammation group. This suggests that these bacteria were introduced during the primary filler injection treatment. The composition of the microbiota on the skin differed markedly from that in the filler, indicating that contamination during the sampling process did not influence results.CONCLUSIONS:Bacteria adherent to soft-tissue fillers or bacteremia probably play a causative role in adverse events. Contamination of samples in the biopsies with skin microbiota was excluded.CLINICAL QUESTION/LEVEL OF EVIDENCE:Therapeutic, III.

Delay in the time-to-positivity of a peripheral blood culture (PBC), the gold standard for early onset neonatal sepsis (EOS) diagnosis, has resulted in excessive use of antibiotics. In this study, we evaluate the potential of the rapid Molecular Culture (MC) assay for quick EOS diagnosis. In the first part of this study, known positive and spiked blood samples were used to assess the performance of MC. In the in vivo clinical study, the second part of this study, all infants receiving antibiotics for suspicion of EOS were included. At initial EOS suspicion, a blood sample was collected for PBC and MC. MC was able to detect bacteria present in the spiked samples even when the bacterial load was low. In the clinical study, MC was positive in one infant with clinical EOS (Enterococcus faecalis) that was not detected by PBC. Additionally, MC was positive in two infants without clinical sepsis (Streptococcus mitis and multiple species), referred to as contamination. The other 37 samples were negative both by MC and PBC. MC seems to be able to detect bacteria even when the bacterial load is low. The majority of MC and PBC results were comparable and the risk for contamination and false positive MC results seems to be limited. Since MC can generate results within 4 h following sampling compared with 36-72 h in PBC, MC may have the potential to replace conventional PBC in EOS diagnostics in order to guide clinicians on when to discontinue antibiotic therapy several hours after birth.

Abstract BACKGROUND AND AIMS Accumulating evidence shows that online post-dilution haemodiafiltration (HDF), especially when a high convection volume is achieved, is associated with a lower mortality risk than haemodialysis (HD) [1]. The mechanism behind this effect, however, is unclear. In this respect, a superior intradialytic organ perfusion and hence, less intestinal tissue damage, permitting the transfer of bacteria from the gut to the blood, might play an important role. Therefore, we assessed whether differences exist in the intradialytic translocation of intact bacteria between four dialysis modalities. METHOD A randomized cross-over trial was performed (NCT03249532, ClinicalTrials.gov) in 11 prevalent dialysis patients, who were exposed to four dialysis strategies: HD with standard dialysate temperature (Td) 36.5°C and cool (Td 35.5°C) dialysate, and HDF with a high (≥23 L/1.73 m2/session) and low (15 L/1.73 m2/session) convection volume. Microbial DNA (mDNA), soluble CD14, high-sensitivity CRP and IL-6 receptor were measured during each modality, as secondary endpoints of this study. Quantitative assessment of mDNA was performed by 16S-23S interspace profiling after DNA isolation [2, 3]. In the current analysis, the quantitative difference in circulating mDNA between modalities was investigated. In addition, differences in the acute phase reaction (APR), as measured by soluble CD14, high-sensitivity CRP and IL-6 receptor and potential relations between mDNA and the APR were analysed. RESULTS DNA of intact bacteria could not be demonstrated in blood samples of the patients in any dialysis strategy. Yet, in all modalities a similar increase in IL-6 receptor, high-sensitivity CRP and soluble CD14 was observed (pre- versus post-dialysis: P-values <.05) (Figures 1A-B). CONCLUSION i) The appearance of DNA from intact bacteria in the blood of dialysis patients could not be demonstrated, despite the use of a sensitive state-of-the-art technique, which is free from contamination by human DNA; ii) during all modalities a comparable and significant APR was observed; iii) hence, the intradialytic APR does not seem to result from translocated intact bacteria.

Selective decontamination of the digestive tract (SDD) is aimed at elimination of potential pathogenic microorganisms. In this study, the effect of SDD on gut microbiota was evaluated in a large homogenous group of elective colorectal cancer surgery patients. Rectal swabs were taken from 118 patients undergoing colorectal surgery. These patients were randomly assigned to receive perioperative SDD or to the control group (no SDD). Rectal swabs were taken prior to surgery, 3 days after commencing administration of SDD. Gut microbial profiles were obtained with the IS-pro technique, a standardized microbiota profiling assay applicable in clinical routine. Differences in abundance for different taxonomical groups and diversity between the groups were assessed. Unsupervised and supervised classification techniques were used to assess microbial signatures, differentiating between the SDD group and the control group. Patients in the SDD group had different gut microbial signatures than in the control group, also in phyla that are not a target for SDD. Escherichia coli, Sutterella spp., Faecalibacterium prausnitzii, and Streptococcus spp. were the species that differed the most between the two groups. The SDD group showed clustering into two subgroups. In one subgroup, a decrease in Proteobacteria was observed, whereas the other subgroup showed a shift in Proteobacteria species. This study shows that SDD not only decreases colonization of the gastrointestinal tract with potential pathogenic Gram-negative microorganisms, but also reduces the abundance of normal colonizers of our gastrointestinal system and leads to a shift in total microbiota composition.