Daniel J. C. Berkhout

Detection of Sepsis in Preterm Infants by Fecal Volatile Organic Compounds Analysis : A Proof of Principle Study

Objectives: Several studies associated altered gut microbiota composition in preterm infants with late-onset sepsis (LOS), up to days before clinical onset of sepsis. Microbiota analysis as early diagnostic biomarker is, however, in clinical practice currently not feasible because of logistic aspects and high costs. Therefore, we hypothesized that analysis of fecal volatile organic compounds (VOCs) may serve as noninvasive biomarker to predict LOS at a preclinical stage, because VOC reflect the composition and activity of intestinal microbial communities. Methods: In a prospective multicenter study, fecal samples were collected daily from infants with a gestational age of <30 weeks. VOC signatures of fecal samples from infants with LOS, collected up to 5 days before diagnosis, were analyzed by means of an electronic nose technology (Cyranose 320) and compared to matched controls. Results: Fecal VOC profiles of infants with LOS (n = 36) could be discriminated from controls (n = 40) at 3 days (area under the curve [±95% confidence interval], P value, sensitivity, specificity; 70.2 [52.2–88.3], 0.033, 57.1%, 61.5%), 2 days (77.7 [62.7–92.7], 0.050, 75.0%, 70.8%), and 1 day (70.4 [49.6–91.3], 0.037, 64.3%, 64.3%) before the onset of LOS. Conclusions: Fecal VOC profiles of preterm infants with LOS could be discriminated from matched controls, up to 3 days before clinical onset of the disease, underlining the hypothesis that intestinal microbiota may play an etiological role in LOS. Notably, VOC profiling is clinically feasible and the potential of this technique in the early detection of LOS needs to be confirmed in future studies.

Sniffing Out Paediatric Gastrointestinal Diseases: The Potential of Volatile Organic Compounds as Biomarkers for Disease

ABSTRACT The diagnostic work-up and follow-up of paediatric functional gastrointestinal disorders and organic conditions usually includes invasive tests, carrying a high burden on patients. There is a place, therefore, for novel, noninvasive disease-specific biomarkers. Volatile organic compounds (VOCs), originating from (patho)physiological metabolic processes in the human body, are excreted as waste products through all conceivable bodily excrements. The spectrum of VOCs harbours a magnificent source of information, with the potential to serve as noninvasive diagnostic biomarkers and to monitor disease activity. VOC analysis has been studied in children and infants with a variety of gastrointestinal diseases, including inflammatory bowel disease, liver diseases, irritable bowel syndrome, necrotizing enterocolitis and infectious diarrhoea. Most of these studies, although limited in sample size, show that patients can be discriminated from controls based on their VOC profiles, underscoring the potential of VOC analysis in diagnosis and follow-up. Currently, however, the application of VOC analysis in clinical practice is limited; substantial challenges, including methodological, biological, and analytical problems, still need to be met. In this review we provide an overview of the available literature on the potential of VOCs as biomarkers for paediatric gastrointestinal diseases. We discuss the available techniques to analyse VOCs and provide topics for VOC-related research, which need to be addressed before VOC diagnostics can be implemented in daily clinical practice.

Development of severe bronchopulmonary dysplasia is associated with alterations in fecal volatile organic compounds

BackgroundThe aim of this study was to evaluate the potential of fecal volatile organic compounds (VOCs), obtained by means of an electronic nose device (Cyranose 320), as early non-invasive biomarker for BPD.MethodsIn this nested case–control study performed at three Neonatal Intensive Care Units, fecal samples obtained at postnatal age of 7, 14, 21, and 28 days from preterm infants with severe bronchopulmonary dysplasia (BPD) were compared with fecal VOC profiles from matched controls. Microbiota analysis was performed by means of IS-pro technique on fecal samples collected at 28 days postnatally.ResultsVOC profiles of infants developing severe BPD (n=15) could be discriminated from matched controls (n=15) at postnatal age of 14 days (area under the curve (±95% confidence interval), P-value, sensitivity, specificity; 0.72 (0.54–0.90), 0.040, 60.0%, 73.3%), 21 days (0.71 (0.52–0.90), 0.049, 66.7%, 73.3%) and 28 days (0.77 (0.59–0.96), 0.017, 69.2%, 69.2%) but not at 7 days. Intestinal microbiota did not differ between BPD subjects and controls.ConclusionFecal VOC profiles of infants developing BPD could be differentiated from controls at postnatal day 14, 21, and 28. VOC differences could not be directed to intestinal microbiota alterations but presumably reflect local and systemic metabolic and inflammatory pathways associated with BPD.

Late-onset Sepsis in Preterm Infants Can Be Detected Preclinically by Fecal Volatile Organic Compound Analysis: A Prospective, Multicenter Cohort Study

Background The intestinal microbiota has increasingly been considered to play a role in the etiology of late-onset sepsis (LOS). We hypothesize that early alterations in fecal volatile organic compounds (VOCs), reflecting intestinal microbiota composition and function, allow for discrimination between infants developing LOS and controls in a preclinical stage. Methods In 9 neonatal intensive care units in the Netherlands and Belgium, fecal samples of preterm infants born at a gestational age ≤30 weeks were collected daily, up to the postnatal age of 28 days. Fecal VOC were measured by high-field asymmetric waveform ion mobility spectrometry (FAIMS). VOC profiles of LOS infants, up to 3 days prior to clinical LOS onset, were compared with profiles from matched controls. Results In total, 843 preterm born infants (gestational age ≤30 weeks) were included. From 127 LOS cases and 127 matched controls, fecal samples were analyzed by means of FAIMS. Fecal VOCs allowed for preclinical discrimination between LOS and control infants. Focusing on individual pathogens, fecal VOCs differed significantly between LOS cases and controls at all predefined time points. Highest accuracy rates were obtained for sepsis caused by Escherichia coli, followed by sepsis caused by Staphylococcus aureus and Staphylococcus epidermidis. Conclusions Fecal VOC analysis allowed for preclinical discrimination between infants developing LOS and matched controls. Early detection of LOS may provide clinicians a window of opportunity for timely initiation of individualized therapeutic strategies aimed at prevention of sepsis, possibly improving LOS-related morbidity and mortality.

Risk Factors for Necrotizing Enterocolitis: A Prospective Multicenter Case-Control Study

Background: The identification of independent clinical risk factors for necrotizing enterocolitis (NEC) may contribute to early selection of infants at risk, allowing for the development of targeted strategies aimed at the prevention of NEC. Objective: The objective of this study was to identify independent risk factors contributing to the development of NEC in a large multicenter cohort. Methods: This prospective cohort study was performed in 9 neonatal intensive care units. Infants born at a gestational age ≤30 weeks were included. Demographic and clinical data were collected daily until day 28 postnatally. Factors predictive of the development of NEC were identified using univariate and multivariable analyses in a 1: 5 matched case-control cohort. Results: In total, 843 infants (56 NEC cases) were included in this study. In the case-control cohort, univariate analysis identified sepsis prior to the onset of NEC and formula feeding to be associated with an increased risk of developing NEC, whereas the administration of antibiotics directly postpartum was inversely associated with NEC. In a multivariable logistic regression model, enteral feeding type and the number of days parenterally fed remained statistically significantly associated with NEC, whereas the administration of antibiotics directly after birth was associated with a lower risk of developing NEC. Conclusions: Formula feeding and prolonged (duration of) parenteral feeding were associated with an increased risk of NEC. Contrary to expectations, the initiation of treatment with antibiotics within 24 h after birth was inversely associated with NEC.

Fecal Volatile Organic Compounds in Preterm Infants Are Influenced by Enteral Feeding Composition

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.

Revival of an ancient Greek art: scent detection as diagnostic tool for tuberculosis

In 2015, the World Health Organization (WHO) included a health target in the newly adopted Sustainable Development Goals, striving to end the tuberculosis (TB) epidemic by the year 2030. In low-income countries, TB continues to be one of the most prevalent infectious diseases with leading mortality rates in the pediatric population. A key reason for these unacceptable high morbidity and mortality rates is the poor sensitivity of available diagnostic tests to detect TB. In these countries, pediatric TB is primarily diagnosed based on clinical signs and smear microscopy, consequently resulting in a large proportion of undiagnosed patients. This illustrates the need for new strategies towards tuberculosis control, including development of new diagnostic modalities. In the interesting article of Mgode et al., the potential of olfactory sense of trained African giant pouched rats (Cricetomys ansorgei) was evaluated as a complementary method in the diagnostic workup of pediatric TB. They showed that implementation of trained rats could increase the detection rate of pediatric TB by 68%, compared to a single-test protocol using smear microscopy. Given the severe health implications of TB, such an increase in detection rate is of considerable clinical importance. Interestingly, already around the year 400 BC, the ancient Greek Hippocrates described the potential of the scent of sputum as diagnostic tool in TB. Over the past years, an increasing number of studies demonstrated the potential of scent molecules, or more specifically volatile organic compounds (VOCs), as a diagnostic biomarker for a wide variety of clinical conditions, including metabolic, infectious, and inflammatory diseases and malignancies. VOCs are carbon-based chemicals, volatile at ambient temperatures, and source of the majority of surrounding odors. Being produced during metabolic processes and excreted through all conceivable bodily excrements (e.g., sputum, feces, sweat, urine), they may serve as an ideal clinical biomarker for several pathophysiological processes. VOCs derived from a particular bodily substrate may either have an endogenous (local or systemic) or exogenous origin. In order to discriminate diseased from healthy state, VOCs derived from different substrates usually provide different outcomes. For example, in intestinal diseases such as inflammatory bowel disease, the discriminative accuracy of fecal VOCs is superior over that of exhaled breath and urinary volatiles. In the study by Mgode et al., sputum was chosen as a substrate of interest, which contains VOCs from both endogenous and exogenous sources. Endogenous VOCs in the sputum include volatiles originating from local pulmonary (patho)physiological processes, whereas systemic VOCs are produced during metabolic processes elsewhere in the body before being transported by the bloodstream towards the alveoli and eventually the sputum. Examples of exogenous sources include resident pulmonary microbes, inhaled VOCs, and medicinal metabolites. In TB patients, sputum presumably contains disease-specific exogenous VOCs derived from the TB-causing pathogens residing in the lungs. Mycobacterium tuberculosis, the most commonly isolated causative agent, is transmitted through aerosol droplets into the alveoli where they enter and proliferate in alveolar innate immune cells. Subsequently, infected cells may form a typical nodular granulomatous structure. In case of uncontrolled growth, this may eventually result in lymphatic and blood vessel invasion, allowing for disease dissemination. Therefore, in addition to exogenous microbial VOCs, the sputum of TB patients may also contain local VOCs derived from pulmonary pathophysiological processes and systemic VOCs derived from other infected tissues. Presumably, presence of TB-specific VOCs with both endogenous and exogenous origins allowed for the differentiation between TB and non-TB cases by giant pouched rats. However, a relatively high false-positive detection rate of approximately 25% by rats was demonstrated in previous studies. Other mycobacterial spp. and non-mycobacterial spp. presumably produce similar VOC profiles, or may provoke comparable hostspecific metabolic reactions, consequently impeding accurate identification of M. tuberculosis in sputum samples. The essential role of both host-specific and pathogen specific VOCs in the identification of TB cases by C. ansorgei is well illustrated in a study in which trained rats were presented TB-negative sputum samples spiked with M. tuberculosis culture isolates and natural TB sputum samples. Rats were able to discriminate the latter samples from controls with higher accuracy than the prepared samples, indicating that the inflammatory host response contributes a TBspecific VOC profile. Several key sputum volatiles, differentiating TB cases from non-TB cases by trained rats, were identified using gas chromatography-mass spectrometry (GC-MS). After isolation of these VOCs they were presented to trained rats. Interestingly, only a particular blend of TB-specific volatiles, provided in measured concentrations, allowed for an adequate reaction from these rats. This illustrates the challenge to unravel and identify the volatiles of interest, evoking the desired detection response in rats. Multiple other studies have attempted to identify TB-specific VOCs using chemical analytical techniques such as GC-MS in a

Smoking influences fecal volatile organic compounds composition

Fecal volatile organic compounds (VOCs) are gaseous carbon-based metabolic products that may serve as diagnostic biomarkers for gastrointestinal diseases, including advanced adenomas, colorectal carcinoma, and inflammatory bowel disease. Fecal VOCs are considered to reflect intestinal microbiota composition, which is influenced by external factors including diet, medication, and smoking. However, data on the influence of smoking on VOC composition are not yet available. Our aim was to assess the effect of smoking on fecal VOC composition.