Host-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants.
ABSTRACT: Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.
Project description:Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease affecting mainly preterm newborns. It is characterized by unexpected onset and rapid progression with specific diagnostic signs as pneumatosis intestinalis or gas in the portal vein appearing later in the course of the disease. Therefore, we analyzed diagnostic and prognostic potential of the markers of early NEC pathogenesis, such as excessive inflammatory response (serum amyloid A (SAA)) and gut epithelium damage (intestinal and liver fatty acid-binding protein (I-FABP and L-FABP, respectively) and trefoil factor-3 (TFF-3)). We used ELISA to analyze these biomarkers in the urine of patients with suspected NEC, either spontaneous or surgery-related, or in infants without gut surgery (controls). Next, we compared their levels with the type of the disease (NEC or sepsis) and its severity. Already at the time of NEC suspicion, infants who developed NEC had significantly higher levels of all tested biomarkers than controls and higher levels of I-FABP and L-FABP than those who will later develop sepsis. Infants who will develop surgery-related NEC had higher levels of I-FABP and L-FABP than those who will develop sepsis already during the first 6 hours after the abdominal surgery. I-FABP was able to discriminate between infants who will develop NEC or sepsis and the SAA was able to discriminate between medical and surgical NEC. Moreover, the combination of TFF-3 with I-FABP and SAA could predict pneumatosis intestinalis, and the combination of I-FABP, L-FABP, and SAA could predict gas in the portal vein or long-term hospitalization and low SAA predicts early full enteral feeding. Thus, these biomarkers may be useful not only in the early, noninvasive diagnostics but also in the subsequent NEC management.
Project description:<h4>Background</h4>Early onset sepsis (EOS) is a major cause of morbidity and mortality in preterm infants, yet diagnosis remains inadequate resulting in missed cases or prolonged empiric antibiotics with adverse consequences. Evaluation of acute phase reactant (APR) biomarkers in umbilical cord blood at birth may improve EOS detection in preterm infants with intrauterine infection.<h4>Methods</h4>In this nested case-control study, infants (29.7 weeks gestation, IQR: 27.7-32.2) were identified from a longitudinal cohort with archived cord blood and placental histopathology. Patients were categorized using culture, laboratory, clinical, and antibiotic treatment data into sepsis groups: confirmed sepsis (cEOS, n = 12); presumed sepsis (PS, n = 30); and no sepsis (controls, n = 30). Nine APRs were measured in duplicate from cord blood using commercially available multiplex immunoassays (Bio-Plex Pro™). In addition, placental histopathologic data were linked to biomarker results.<h4>Results</h4>cEOS organisms were Escherichia coli, Streptococcus agalactiae, Proteus mirabilis, Haemophilus influenzae and Listeria monocytogenes. C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), serum amyloid P and ferritin were significantly elevated in cEOS compared to controls (p<0.01). SAA, CRP, and Hp were elevated in cEOS but not in PS (p<0.01) and had AUCs of 99%, 96%, and 95% respectively in predicting cEOS. Regression analysis revealed robust associations of SAA, CRP, and Hp with EOS after adjustment for covariates. Procalcitonin, fibrinogen, ?-2-macroglobulin and tissue plasminogen activator were not significantly different across groups. Placental acute inflammation was associated with APR elevation and was present in all cEOS, 9 PS, and 17 control infants.<h4>Conclusion</h4>This study shows that certain APRs are elevated in cord blood of premature infants with EOS of intrauterine origin. SAA, CRP, and Hp at birth have potential diagnostic utility for risk stratification and identification of infants with EOS.
Project description:We aim to determine risk factors and clinical outcomes for bowel perforation in premature infants with NEC. We analyzed clinical data of 57 cases of premature infants with NEC at our NICU between January 2010 and December 2012. Based on the presence of bowel perforation, we divided these infants into two groups: perforated NEC group (n = 10) and nonperforated NEC group (n = 47). We compared general information, clinical characteristics, and laboratory findings between groups. The perforated NEC group, compared to the nonperforated NEC group, had significantly lesser gestational age, lower birth weight, higher prevalence of apnea, mechanical ventilation, sepsis and shock, lower blood pH, higher levels of blood glucose, abnormal WBC count and thrombocytopenia, and elevated CRP (all P < 0.05). Moreover, the perforated NEC group had significantly longer durations of fasting and TPN usage, higher incidences of EUGR and cholestasis, longer duration of antibiotics, higher frequency of advanced antibiotics use, and poorer prognosis than the nonperforated NEC group (all P < 0.05). Bowel perforation in premature infants with NEC was associated with multiple risk factors. Early identification of some of these risk factors in premature infants with NEC may help implement early intervention to reduce the incidence of bowel perforation and thereby improve the prognosis.
Project description:The assembly and development of the gut microbiome in infants have important consequences for immediate and long-term health. Preterm infants represent an abnormal case for bacterial colonization because of early exposure to bacteria and frequent use of antibiotics. To better understand the assembly of the gut microbiota in preterm infants, fecal samples were collected from 32 very low birth weight preterm infants over the first 6 weeks of life. Infant health outcomes included health, late-onset sepsis, and necrotizing enterocolitis (NEC). We characterized bacterial compositions by 16S rRNA gene sequencing and metabolomes by untargeted gas chromatography-mass spectrometry. Preterm infant fecal samples lacked beneficial Bifidobacterium spp. and were dominated by Enterobacteriaceae, Enterococcus, and Staphylococcus organisms due to nearly uniform antibiotic administration. Most of the variance between the microbial community compositions could be attributed to the baby from which the sample derived (permutational multivariate analysis of variance [PERMANOVA] R2 = 0.48, P < 0.001), while clinical status (health, NEC, or late-onset sepsis) and overlapping times in the neonatal intensive care unit (NICU) did not explain a significant amount of variation in bacterial composition. Fecal metabolomes were also found to be unique to the individual (PERMANOVA R2 = 0.43, P < 0.001) and weakly associated with bacterial composition (Mantel statistic r = 0.23 ± 0.05, P < 0.05). No measured metabolites were found to be associated with necrotizing enterocolitis, late-onset sepsis, or a healthy outcome. Overall, preterm infant gut microbial communities were personalized and reflected antibiotic usage.IMPORTANCE Preterm infants face health problems likely related to microbial exposures, including sepsis and necrotizing enterocolitis. However, the role of the gut microbiome in preterm infant health is poorly understood. Microbial colonization differs from that of healthy term babies because it occurs in the NICU and is often perturbed by antibiotics. We measured bacterial compositions and metabolomic profiles of 77 fecal samples from 32 preterm infants to investigate the differences between microbiomes in health and disease. Rather than finding microbial signatures of disease, we found that both the preterm infant microbiome and the metabolome were personalized and that the preterm infant gut microbiome is enriched in microbes that commonly dominate in the presence of antibiotics. These results contribute to the growing knowledge of the preterm infant microbiome and emphasize that a personalized view will be important to disentangle the health consequences of the preterm infant microbiome.
Project description:Background: The development of necrotizing enterocolitis (NEC) in neonates with patent ductus arteriosus (PDA) is not well-understood. Our aim was to find risk factors for NEC in children with a significant PDA and to assess differences in mortality and duration of hospital stay between patients with PDA and those with PDA and NEC. Methods: We performed a retrospective single center case control study including infants with PDA scheduled for treatment. We compared multiple patient data between patients with PDA and those with PDA and NEC from 2004 to 2018 using 1:2 and 1:1 matching. Results: We used 1:2 matching with 26 NEC patients (cases) and 52 PDA patients without NEC (controls) and 1:1 matching with 5 NEC patients and 5 PDA patients without NEC. NEC patients had lower Apgar score (1'), more congenital malformations, more suspected sepsis, less hypotension, higher minimum platelet count and higher CRP-values during the week before NEC (P < 0.05, respectively). The mortality was higher in NEC cases [29% (9/31)] compared to the control patients [2% (1/57), P < 0.001]. Lower Apgar score (1') was correlated with an increased risk of NEC stage III. Hypotension was inversely correlated with the odds of NEC (OR 0.3). Conclusions: NEC increased mortality in infants with PDA. Hypotension did not increase the risk of NEC in infants with PDA. Routine clinical parameters were not able to predict NEC in infants who suffer from PDA.
Project description:<h4>Objective</h4>To investigate secretor gene fucosyltransferase 2 (FUT2) polymorphism and secretor phenotype in relation to outcomes of prematurity.<h4>Study design</h4>Study infants were ?32 weeks gestational age. Secretor genotype was determined from salivary DNA. Secretor phenotype was measured with H antigen, the carbohydrate produced by secretor gene enzymes, in saliva samples collected on day 9 ± 5. The optimal predictive cutoff point in salivary H values was identified with Classification and Regression Tree analysis. Study outcomes were death, necrotizing enterocolitis (NEC, Bell's stage II/III), and confirmed sepsis.<h4>Results</h4>There were 410 study infants, 26 deaths, 30 cases of NEC, and 96 cases of sepsis. Analyzed by genotype, 13% of 95 infants who were non-secretors, 5% of 203 infants who were heterozygotes, and 2% of 96 infants who were secretor dominant died (P = .01). Analyzed by phenotype, 15% of 135 infants with low secretor phenotype died, compared with 2% of 248 infants with high secretor phenotype (predictive value = 76%, P < .001). Low secretor phenotype was associated (P < .05) with NEC, and non-secretor genotype was associated (P = .05) with gram negative sepsis. Secretor status remained significant after controlling for multiple clinical factors.<h4>Conclusions</h4>Secretor genotype and phenotype may provide strong predictive biomarkers of adverse outcomes in premature infants.
Project description:Importance:Necrotizing enterocolitis (NEC) in preterm infants is an often-fatal gastrointestinal tract emergency. A robust NEC biomarker that is not confounded by sepsis could improve bedside management, lead to lower morbidity and mortality, and permit patient selection in randomized clinical trials of possible therapeutic approaches. Objective:To evaluate whether aberrant intestinal alkaline phosphatase (IAP) biochemistry in infant stool is a molecular biomarker for NEC and not associated with sepsis. Design, Setting, and Participants:This multicenter diagnostic study enrolled 136 premature infants (gestational age, <37 weeks) in 2 hospitals in Louisiana and 1 hospital in Missouri. Data were collected and analyzed from May 2015 to November 2018. Exposures:Infant stool samples were collected between 24 and 40 or more weeks postconceptual age. Enrolled infants underwent abdominal radiography at physician and hospital site discretion. Main Outcomes and Measures:Enzyme activity and relative abundance of IAP were measured using fluorometric detection and immunoassays, respectively. After measurements were performed, biochemical data were evaluated against clinical entries from infants' hospital stay. Results:Of 136 infants, 68 (50.0%) were male infants, median (interquartile range [IQR]) birth weight was 1050 (790-1350) g, and median (IQR) gestational age was 28.4 (26.0-30.9) weeks. A total of 25 infants (18.4%) were diagnosed with severe NEC, 19 (14.0%) were suspected of having NEC, and 92 (66.9%) did not have NEC; 26 patients (19.1%) were diagnosed with late-onset sepsis, and 14 (10.3%) had other non-gastrointestinal tract infections. For severe NEC, suspected NEC, and no NEC samples, median (IQR) fecal IAP content, relative to the amount of IAP in human small intestinal lysate, was 99.0% (51.0%-187.8%) (95% CI, 54.0%-163.0%), 123.0% (31.0%-224.0%) (95% CI, 31.0%-224.0%), and 4.8% (2.4%-9.8%) (95% CI, 3.4%-5.9%), respectively. For severe NEC, suspected NEC, and no NEC samples, median (IQR) enzyme activity was 183 (56-507) ?mol/min/g (95% CI, 63-478 ?mol/min/g) of stool protein, 355 (172-608) ?mol/min/g (95% CI, 172-608 ?mol/min/g) of stool protein, and 613 (210-1465) ?mol/min/g (95% CI, 386-723 ?mol/min/g) of stool protein, respectively. Mean (SE) area under the receiver operating characteristic curve values for IAP content measurements were 0.97 (0.02) (95% CI, 0.93-1.00; P?<?.001) at time of severe NEC, 0.97 (0.02) (95% CI, 0.93-1.00; P?<?.001) at time of suspected NEC, 0.52 (0.07) (95% CI, 0.38-0.66; P?=?.75) at time of sepsis, and 0.58 (0.08) (95% CI, 0.42-0.75; P?=?.06) at time of other non-gastrointestinal tract infections. Mean (SE) area under the receiver operating characteristic curve values for IAP activity were 0.76 (0.06) (95% CI, 0.64-0.86; P?<?.001), 0.62 (0.07) (95% CI, 0.48-0.77; P?=?.13), 0.52 (0.07) (95% CI, 0.39-0.67; P?=?.68), and 0.57 (0.08) (95% CI, 0.39-0.69; P?=?.66), respectively. Conclusions and Relevance:In this diagnostic study, high amounts of IAP protein in stool and low IAP enzyme activity were associated with diagnosis of NEC and may serve as useful biomarkers for NEC. Our findings indicated that IAP biochemistry was uniquely able to distinguish NEC from sepsis.
Project description:Preterm infants are highly susceptible to late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) but specific biomarkers for diagnosis and effective treatment are lacking. Neutrophil extracellular traps (NETs) are related to sepsis in adults but not investigated in infant conditions. This is the first proteome study to document that circulating NETs are involved in neonatal LOS and NEC. cfDNA and NET proteins may provide new potential diagnostic markers for these diseases.
Project description:Preterm birth, bacterial colonization, and formula feeding predispose to necrotizing enterocolitis (NEC). Antibiotics are commonly administered to prevent sepsis in preterm infants, but it is not known whether this affects intestinal immunity and NEC resistance. We hypothesized that broad-spectrum antibiotic treatment improves NEC resistance and intestinal structure, function, and immunity in neonates. Caesarean-delivered preterm pigs were fed 3 days of parenteral nutrition followed by 2 days of enteral formula. Immediately after birth, they were assigned to receive either antibiotics (oral and parenteral doses of gentamycin, ampicillin, and metronidazole, ANTI, n = 11) or saline in the control group (CON, n = 13), given twice daily. NEC lesions and intestinal structure, function, microbiology, and immunity markers were recorded. None of the ANTI but 85% of the CON pigs developed NEC lesions by day 5 (0/11 vs. 11/13, P < 0.05). ANTI pigs had higher intestinal villi (+60%), digestive enzyme activities (+53-73%), and goblet cell densities (+110%) and lower myeloperoxidase (-51%) and colonic microbial density (10(5) vs. 10(10) colony-forming units, all P < 0.05). Microarray transcriptomics showed strong downregulation of genes related to inflammation and innate immune response to microbiota and marked upregulation of genes related to amino acid metabolism, in particular threonine, glucose transport systems, and cell cycle in 5-day-old ANTI pigs. In a follow-up experiment, 5 days of antibiotics prevented NEC at least until day 10. Neonatal prophylactic antibiotics effectively reduced gut bacterial load, prevented NEC, intestinal atrophy, dysfunction, and inflammation and enhanced expression of genes related to gut metabolism and immunity in preterm pigs.
Project description:BACKGROUND:Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants. METHODS:Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization-time of flight. Clostridial isolates were typed and toxin genes detected. RESULTS:A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk. CONCLUSIONS:Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC. Clinical Trials Registration.?NCT01102738.