Background: Late-onset sepsis (LOS) in preterm infants is a leading cause of mortality and morbidity. Timely recognition and initiation of antibiotics are important factors for improved outcomes. Identification of risk factors could allow selection of infants at an increased risk for LOS. Objectives: The aim was to identify risk factors for LOS. Methods: In this multicenter case-control study, preterm infants born at ≤30 weeks of gestation were included at 9 neonatal intensive care units. Detailed demographical and clinical data were collected daily up to day 28 postnatally. Clinical and demographic risk factors were identified using univariate and multivariate regression analyses in a 1: 1 matched case-control cohort. Results: In total, 755 infants were included, including 194 LOS cases (41 gram-negative cases, 152 gram-positive cases, and 1 fungus). In the case-control cohort, every additional day of parenteral feeding increased the risk for LOS (adjusted OR = 1.29; 95% CI 1.07–1.55; p = 0.006), whereas antibiotics administration decreased this risk (OR = 0.08; 95% CI 0.01–0.88; p = 0.039). These findings could largely be attributed to specific LOS-causative pathogens, since these predictive factors could be identified for gram-positive, but not for gram-negative, LOS cases. Specifically cephalosporins administration prior to clinical onset was inversely related to coagulase-negative staphylococcus LOS (CoNS-LOS) development. Formula feeding was an independent risk factor for development of CoNS-LOS (OR = 3.779; 95% CI 1.257–11.363; p = 0.018). Conclusion: The length of parenteral feeding was associated with LOS, whereas breastmilk administration was protective against CoNS-LOS. A rapid advancement of enteral feeding, preferably with breastmilk, may proportionally reduce the number of parenteral feeding days and consequently the risk for LOS.

Fecal volatile organic compounds (VOCs) are increasingly considered to be potential noninvasive, diagnostic biomarkers for various gastrointestinal diseases. Knowledge of the influence of sampling conditions on VOC outcomes is limited. We aimed to evaluate the effects of sampling conditions on fecal VOC profiles and to assess under which conditions an optimal diagnostic accuracy in the discrimination between pediatric inflammatory bowel disease (IBD) and controls could be obtained. Fecal samples from de novo treatment-naïve pediatric IBD patients and healthy controls (HC) were used to assess the effects of sampling conditions compared to the standard operating procedure (reference standard), defined as 500 mg of sample mass diluted with 10 mL tap water, using field asymmetric ion mobility spectrometry (FAIMS). A total of 17 IBD (15 CD (Crohn's disease) and 2 UC (ulcerative colitis)) and 25 HC were included. IBD and HC could be discriminated with high accuracy (accuracy = 0.93, AUC = 0.99, p < 0.0001). A smaller fecal sample mass resulted in a decreased diagnostic accuracy (300 mg accuracy = 0.77, AUC = 0.69, p = 0.02; 100 mg accuracy = 0.70, AUC = 0.74, p = 0.003). A loss of diagnostic accuracy was seen toward increased numbers of thaw–freeze cycles (one cycle, accuracy = 0.61, AUC = 0.80, p = 0.0004; two cycles, accuracy = 0.64, AUC = 0.56, p = 0.753; and three cycles, accuracy = 0.57, AUC = 0.50, p = 0.5101) and when samples were kept at room temperature for 180 min prior to analysis (accuracy = 0.60, AUC = 0.51, p = 0.46). Diagnostic accuracy of VOC profiles was not significantly influenced by storage duration differences of 20 months. The application of a 500 mg sample mass analyzed after one thaw–freeze cycle showed the best discriminative accuracy for the differentiation of IBD and HC. VOC profiles and diagnostic accuracy were significantly affected by sampling conditions, underlining the need for the implementation of standardized protocols in fecal VOC analysis.

Fecal volatile organic compound (VOC) analysis has shown great potential as a noninvasive diagnostic biomarker for a variety of diseases. Before clinical implementation, the factors influencing the outcome of VOC analysis need to be assessed. Recent studies found that the sampling conditions can influence the outcome of VOC analysis. However, the dietary influences remains unknown, especially in (preterm) infants. Therefore, we assessed the effects of feeding composition on fecal VOC patterns of preterm infants (born at <30 weeks gestation). Two subgroups were defined: (1) daily intake >75% breastmilk (BM) feeding and (2) daily intake >75% formula milk (FM) feeding. Fecal samples, which were collected at 7, 14 and 21 days postnatally, were analyzed by an electronic nose device (Cyranose 320®). In total, 30 preterm infants were included (15 FM, 15 BM). No differences in the fecal VOC patterns were observed at the three predefined time-points. Combining the fecal VOC profiles of these time-points resulted in a statistically significant difference between the two subgroups although this discriminative accuracy was only modest (AUC [95% CI]; p-value; sensitivity; and specificity of 0.64 [0.51–0.77]; 0.04; 68%; and 51%, respectively). Our results suggest that the influence of enteral feeding on the outcome of fecal VOC analysis cannot be ignored in this population. Furthermore, in both subgroups, the fecal VOC patterns showed a stable longitudinal course within the first month of life.

Background The role of intestinal microbiota in the pathogenesis of late onset sepsis (LOS) in preterm infants is largely unexplored, but could provide opportunities for microbiota-targeted preventive and therapeutic strategies. We hypothesized that microbiota composition changes before the onset of sepsis with causative bacteria that are isolated later in blood culture. Methods In this multicenter case-control study preterm infants born under 30 weeks of gestation were included. Fecal samples collected from the five days preceding LOS diagnosis, were analyzed by a molecular microbiota detection technique. LOS cases were subdivided into three groups: Gram-negative, Gram-positive and Coagulase negative Staphylococci (CoNS). Results Totally, 40 LOS cases and 40 matched controls were included. In Gram-negative LOS, the causative pathogen could be identified in at least one of the fecal samples collected three days prior LOS onset in all cases, whereas in all matched controls this pathogen was absent (p-value 0.015). The abundance of these pathogens increased from three days towards clinical onset. In Gram-negative and -positive LOS (except CoNS) combined, the causative pathogen could be identified in at least one fecal sample collected three days prior to LOS onset in 92% of the fecal samples of cases, whereas these pathogens were present in 33% of the control samples (p-value 0.004). Overall, LOS (expect CoNS) could be predicted one day prior clinical onset with an AUC of 0.78. Conclusions Profound preclinical microbial alterations underline that gut microbiota is involved in the pathogenesis of LOS and has the potential as early non-invasive biomarker.

Emerging evidence exists that an altered gut microbiota is a key factor in the pathophysiology of a variety of diseases. Consequently, microbiota-targeted interventions, including administration of probiotics, have increasingly been evaluated. Mechanisms on how probiotics contribute to homeostasis or reverse (effects of) dysbiosis remain yet to be elucidated. In the current study, we assessed the effects of daily Lactobacillus casei strain Shirota (LcS) ingestion in healthy children aged from 12–18 years on gut microbiota compositional diversity and stability. Results were compared to healthy children without LcS exposure. For a period of 6 weeks, fecal samples were collected weekly by both groups. In total, 18 children were included (6 probiotics; 12 non-probiotics). At 1-week intervals, no differences in diversity and stability were observed in children exposed to LcS versus controls. LcS ingestion by healthy children does not result in a more diverse and stable gut microbiota composition. Large double-blind placebo-controlled randomized clinical trials in children should be performed to gain more insight on potential beneficial health consequences.

Necrotizing enterocolitis (NEC) is one of the most common and lethal gastrointestinal diseases in preterm infants. Early recognition of infants in need for surgical intervention might enable early intervention. In this multicenter case-control study, performed in nine neonatal intensive care units, preterm born infants (< 30 weeks of gestation) diagnosed with NEC (stage ≥ IIA) between October 2014 and August 2017 were divided into two groups: (1) medical (conservative treatment) and (2) surgical NEC (sNEC). Perinatal, clinical, and laboratory parameters were collected daily up to clinical onset of NEC. Univariate and multivariate logistic regression analyses were applied to identify potential predictors for sNEC. In total, 73 preterm infants with NEC (41 surgical and 32 medical NEC) were included. A low gestational age (p value, adjusted odds ratio [95%CI]; 0.001, 0.91 [0.86–0.96]), no maternal corticosteroid administration (0.025, 0.19 [0.04–0.82]), early onset of NEC (0.003, 0.85 [0.77–0.95]), low serum bicarbonate (0.009, 0.85 [0.76–0.96]), and a hemodynamically significant patent ductus arteriosus for which ibuprofen was administered (0.003, 7.60 [2.03–28.47]) were identified as independent risk factors for sNEC.Conclusions: Our findings may support the clinician to identify infants with increased risk for sNEC, which may facilitate early decisive management and consequently could result in improved prognosis. What is Known:• In 27–52% of the infants with NEC, a surgical intervention is indicated during its disease course.• Absolute indication for surgical intervention is bowel perforation, whereas fixed bowel loop or clinical deterioration highly suggestive of bowel perforation or necrosi, is a relative indication. What is New:• Lower gestational age, early clinical onset, and no maternal corticosteroids administration are predictors for surgical NEC.• Low serum bicarbonate in the 3 days prior clinical onset and patent ductus arteriosus for which ibuprofen was administered predict surgical NEC.

Abstract:Increasing interest is noticed in the potential of volatile organic compound (VOC) analysis as non-invasive diagnostic biomarker in clinical medical practice. The spectrum of VOCs, originating from (patho)physiological metabolic processes in the human body and detectable in bodily excrements, such as exhaled breath, urine and feces, harbors a magnificent source of information. Thus far, the majority of studies have focused on VOC analysis in exhaled breath, aiming at identification of disease-specific VOC profiles. Recently, an increasing number of studies have evaluated the usability of VOC present in the headspace of feces in the diagnostic work-up of a wide range of gastrointestinal diseases. Promising results have been demonstrated particularly in those diseases in which microbiota alterations are considered to play a significant etiological role, such as colorectal carcinoma, inflammatory bowel disease, irritable bowel syndrome, celiac disease and infectious bowel diseases. In addition, fecal VOC analysis seems to have potential as a diagnostic biomarker for extra-intestinal diseases, including bronchopulmonary dysplasia and sepsis. Different methods for VOC analysis have been used in medical studies, such as gas-chromatography mass spectrometry, selected-ion flow tube-mass spectrometry, ion-mobility spectrometry, and electronic nose devices. In this review, the available literature on the potential of fecal VOCs as diagnostic biomarker, including an overview of relevant VOC detection techniques, is discussed. In addition, future hurdles, which need to be taken prior to implementation of VOC analysis in daily clinical practice, are outlined.

Introduction Headspace gas chromatography–mass spectrometry (HS-GC–MS) is widely considered the gold standard of quantitative fecal VOC analysis. However, guidelines providing general recommendations for bioanalytical method application in research and clinical setting are lacking. Objectives To propose an evidence-based research protocol for fecal VOC analysis by HS-GC–MS, based on extensive testing of instrumental and sampling conditions on detection and quantification limits, linearity, accuracy and repeatability of VOC outcome. Methods The influence of the following variables were assessed: addition of different salt solutions, injection temperature, injection speed, injection volume, septum use, use of calibration curves and fecal sample mass. Ultimately, the optimal sample preparation was assessed using fecal samples from healthy preterm infants. Fecal VOC analysis in this specific population has potential as diagnostic biomarkers, but available amount of feces is limited here, so optimization of VOC extraction is of importance. Results We demonstrated that addition of lithium chloride enhanced the release of polar compounds (e.g. small alcohols) into the headspace. Second, a linear relationship between injection volume, speed and temperature, and fecal sample mass on the abundance of VOC was demonstrated. Furthermore, the use of a septum preserved 90% of the non-polar compounds. By application of optimal instrumental and sampling conditions, a maximum of 320 unique compounds consisting of 14 different chemical classes could be detected. Conclusions These findings may contribute to standardized analysis of fecal VOC by HS-GC–MS, facilitating future application of fecal VOC in clinical practice.