Universal Digital High-Resolution Melt Analysis for the Diagnosis of Bacteremia.

Fast and accurate diagnosis of bloodstream infection is necessary to inform treatment decisions for septic patients, who face hourly increases in mortality risk. Blood culture remains the gold standard test but typically requires approximately 15 hours to detect the presence of a pathogen. We, therefore, assessed the potential for universal digital high-resolution melt (U-dHRM) analysis to accomplish faster broad-based bacterial detection, load quantification, and species-level identification directly from whole blood. Analytical validation studies demonstrated strong agreement between U-dHRM load measurement and quantitative blood culture, indicating that U-dHRM detection is highly specific to intact organisms. In a pilot clinical study of 17 whole blood samples from pediatric patients undergoing simultaneous blood culture testing, U-dHRM achieved 100% concordance when compared with blood culture and 88% concordance when compared with clinical adjudication. Moreover, U-dHRM identified the causative pathogen to the species level in all cases where the organism was represented in the melt curve database. These results were achieved with a 1-mL sample input and sample-to-answer time of 6 hours. Overall, this pilot study suggests that U-dHRM may be a promising method to address the challenges of quickly and accurately diagnosing a bloodstream infection.

Decoding human cytomegalovirus for the development of innovative diagnostics to detect congenital infection.

Cytomegalovirus is the most common cause of congenital infectious disease and the leading nongenetic etiology of sensorineural hearing loss. Although most infected neonates are asymptomatic at birth, congenital cytomegalovirus infection is responsible for nearly 400 infant deaths annually in the United States and may lead to significant long-term neurodevelopmental impairments in survivors. The resulting financial and social burdens of congenital cytomegalovirus infection have led many medical centers to initiate targeted testing after birth, with a growing advocacy to advance universal newborn screening. While no cures or vaccines are currently available to eliminate or prevent cytomegalovirus infection, much has been learned over the last five years regarding disease pathophysiology and viral replication cycles that may enable the development of innovative diagnostics and therapeutics. This Review will detail our current understanding of congenital cytomegalovirus infection, while focusing our discussion on routine and emerging diagnostics for viral detection, quantification, and long-term prognostication. IMPACT: This review highlights our current understanding of the fetal transmission of human cytomegalovirus. It details clinical signs and physical findings of congenital cytomegalovirus infection. This submission discusses currently available cytomegalovirus diagnostics and introduces emerging platforms that promise improved sensitivity, specificity, limit of detection, viral quantification, detection of genomic antiviral resistance, and infection staging (primary, latency, reactivation, reinfection).

A broad-based probe-free qPCR assay for detection and discrimination of three human herpes viruses.

Primary infection or reactivation of latent human cytomegalovirus (HCMV) or herpes simplex viruses (HSV) 1 or 2 during pregnancy can transmit the virus in utero or during natural childbirth to the fetus. The majority of these infections are asymptomatic at birth but may present later with potentially lethal disseminated infection or meningitis (HSV), or long-term neurodevelopmental sequelae including sensorineural hearing loss or neurodevelopmental impairments (HCMV). Unfortunately, early signs and symptoms of disseminated viral infections may be misdiagnosed as bacterial sepsis. Therefore, immediate testing for viral etiologies may not be ordered or even considered by skilled clinicians. In asymptomatic HCMV infections, early detection is necessary to monitor for and treat future neurologic sequelae. In acutely ill-appearing infants, specific detection of viruses against other disease-causing agents is vital to inform correct patient management, including early administration of the correct antimicrobial(s). An ideal test should be rapid, inexpensive, require low sample volumes, and demonstrate efficacy in multiple tissue matrices to aid in timely clinical decision-making for neonatal infections. This work discusses the development of a rapid probe-free qPCR assay for HSV and HCMV that enables early and specific detection of these viruses in neonates. The assay's probe free chemistry would allow easier extension to a broad-based multiplexed pathogenic panel as compared to assays utilizing sequence-specific probes or nested PCR.

Universal digital high resolution melt analysis for the diagnosis of bacteremia.

Fast and accurate diagnosis of bloodstream infection is necessary to inform treatment decisions for septic patients, who face hourly increases in mortality risk. Blood culture remains the gold standard test but typically requires ∼15 hours to detect the presence of a pathogen. Here, we assess the potential for universal digital high-resolution melt (U-dHRM) analysis to accomplish faster broad-based bacterial detection, load quantification, and species-level identification directly from whole blood. Analytical validation studies demonstrated strong agreement between U-dHRM load measurement and quantitative blood culture, indicating that U-dHRM detection is highly specific to intact organisms. In a pilot clinical study of 21 whole blood samples from pediatric patients undergoing simultaneous blood culture testing, U-dHRM achieved 100% concordance when compared with blood culture and 90.5% concordance when compared with clinical adjudication. Moreover, U-dHRM identified the causative pathogen to the species level in all cases where the organism was represented in the melt curve database. These results were achieved with a 1 mL sample input and sample-to-answer time of 6 hrs. Overall, this pilot study suggests that U-dHRM may be a promising method to address the challenges of quickly and accurately diagnosing a bloodstream infection. Universal digital high resolution melt analysis for the diagnosis of bacteremia: April Aralar, Tyler Goshia, Nanda Ramchandar, Shelley M. Lawrence, Aparajita Karmakar, Ankit Sharma, Mridu Sinha, David Pride, Peiting Kuo, Khrissa Lecrone, Megan Chiu, Karen Mestan, Eniko Sajti, Michelle Vanderpool, Sarah Lazar, Melanie Crabtree, Yordanos Tesfai, Stephanie I. Fraley.

Severe Anemia at Birth-Incidence and Implications.

OBJECTIVE: To identify neonates with severe anemia at birth, defined by a hemoglobin or hematocrit value within the first 6 hours after birth that plotted below the 1st percentile according to gestational age. For each patient, we retrospectively determined whether caregivers recognized the anemia within the first 24 hours after birth and the probable cause and outcome of anemia. STUDY DESIGN: This was a retrospective cohort analysis of Intermountain Healthcare population-based data from neonates born between January 2011 and December 2020 who had a hemoglobin or hematocrit value measured within the first 6 hours after birth below the 1st percentile lower reference interval (hematocrit ∼35% in near-term/term neonates). RESULT: Among 299 927 live births, we identified 344 neonates with severe anemia at birth. In 191 of these neonates (55.5%), the anemia was recognized by caregivers during the first 24 hours. Anemia was more likely to be recorded as a problem (85%) if the hemoglobin was ≥2 g/dL below the 1st percentile (P < .001). The lowest hemoglobin values occurred in those in whom hemorrhage was the probable cause (P < .013 vs hemolysis and P < .001 vs hypoproduction, mixed cause, or indeterminant.) Treatment was provided to 39.5%. A retrospective review suggested that mixed mechanisms, particularly hemorrhagic plus hemolytic, occurred more commonly than was recognized at the time of occurrence. CONCLUSIONS: Severe anemia at birth often went unrecognized on the first day of life. Algorithm-directed retrospective reviews commonly identified causes that were not listed in the medical record. We postulate that earlier recognition and more accurate diagnoses would be facilitated by an electronic medical record-associated hemoglobin/hematocrit gestational age nomogram.

Implications of an Elevated Nucleated Red Blood Cell Count in Neonates with Moderate to Severe Hypoxic-Ischemic Encephalopathy.

OBJECTIVES: To investigate associations between nucleated red blood cell (NRBC) count in neonates with hypoxic-ischemic encephalopathy (HIE), acute perinatal sentinel events, and neurodevelopmental outcomes and to examine the mechanism(s) causing elevated counts. STUDY DESIGN: We included newborn infants with HIE treated with therapeutic hypothermia with ≥3 NRBC counts during their neonatal intensive care unit hospitalization and neurodevelopmental evaluations at a mean of 24 ± 6 months. RESULTS: Ninety-five of 152 infants who met our study criteria (63%) had a normal NRBC count after birth, defined as ≤95th percentile of the upper reference interval, and the other 57 (37%) had an elevated count. Documented sentinel events during labor resulting in emergency delivery (eg, acute abruption) (n = 79) were associated with a normal NRBC count (OR, 257; 95% CI, 33-1988). Of the 152 infants evaluated, 134 (88%) survived to discharge. The odds of surviving were 3-fold greater (OR, 3.0; 95% CI, 1.1-8.3) when the first NRBC count was normal than when it was elevated. Normal counts were moderately predictive of infants without neurodevelopmental impairment at a 2-year evaluation (P < .001). NRBC half-life was longer in infants with an elevated NRBC count compared with those with a normal count (60 hours vs 39 hours; P < .01). CONCLUSIONS: In infants with HIE, a normal NRBC count after birth was associated with acute intrapartum events necessitating emergent delivery. Normal counts were modestly predictive of a better prognosis. We speculate that the elevated NRBC counts at birth resulted from hypoxia that occurred earlier or chronically. Impaired clearance of NRBCs from the blood might be one mechanistic explanation for the high counts.

Perinatal Carbon Monoxide Poisoning: Treatment of a 2-Hour-Old Neonate with Hyperbaric Oxygen.

A 41-year-old gravida 4 para 3 (G4P3) and 38 5/7 weeks pregnant woman presented to labor and delivery with dizziness, headache, and decreased fetal movement after 12 hours of exposure to carbon monoxide (CO) from a grill that was used inside for heat. The mother was hemodynamically stable, and her neurologic examination was intact. Her carboxyhemoglobin level, which was obtained 12 hours after removal from the CO exposure, was 7.4%. The fetus's heart rate was 173 beats per minute with moderate variability and one late appearing deceleration, not associated with contractions. The biophysical profile score was 2 of 8. The obstetrics team performed a routine cesarean section. The 1- and 5-minute Apgar's scores were 7 and 8, respectively. The arterial cord gas result was as follows: pH = 7.05, PCO 2 = 71 mm Hg, pO 2 = 19 mm Hg, bicarbonate = 14 mmol/L, and carboxyhemoglobin = 11.9%. The mother and infant were treated with hyperbaric oxygen therapy consisting of 100% oxygen at 2.4 atmosphere absolutes (ATA) for 90 minutes at 2.5 hours after delivery. Following one hyperbaric oxygen treatment, the infant was transitioned to room air and routine postpartum treatment and was discharged 3 days later in good condition. Hyperbaric oxygen treatment was well tolerated in this neonate.

Contribution of Concurrent Comorbidities to Sepsis-Related Mortality in Preterm Infants ≤32 Weeks of Gestation at an Academic Neonatal Intensive Care Network.

OBJECTIVE: This study sought to identify concurrent major comorbidities in preterm infants ≤32 weeks of gestation that may have contributed to sepsis-related mortality following a diagnosis of bacteremia or blood culture-negative sepsis within the neonatal period (≤28 days of life). STUDY DESIGN: This is a retrospective chart review of infants ≤32 weeks of gestation who were admitted to a single academic network of multiple neonatal intensive care units between January 1, 2012, and December 31, 2015, to determine the primary cause(s) and timing of death in those diagnosed with bacteremia or blood culture-negative sepsis. Direct comparisons between early-onset sepsis (EOS; ≤72 hours) and late-onset sepsis (LOS; >72 hours) were made. RESULTS: In our study cohort, of 939 total patients with ≤32 weeks of gestation, 182 infants were diagnosed with 198 episodes of sepsis and 7.7% (14/182) died. Mortality rates did not significantly differ between neonates with bacteremia or blood culture-negative sepsis (7/14 each group), and those diagnosed with EOS compared with LOS (6/14 vs. 8/14). Nearly 80% (11/14) of infants were transitioned to comfort care prior to their death secondary to a coinciding diagnosis of severe grade-3 or -4 intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, and/or intestinal perforation. CONCLUSION: Preexisting comorbidities commonly associated with extreme preterm birth contributed to sepsis-related mortality in our patient cohort. KEY POINTS: · Concurrent comorbidities contribute to, and may artificially inflate, sepsis-related mortality.. · Absence of a consensus definition for neonatal sepsis complicates the investigation of infection.. · Accurate assessment of the incidence of sepsis in very low birth weight infants is vital for future investigations.

Data-driven noise modeling of digital DNA melting analysis enables prediction of sequence discriminating power.

MOTIVATION: The need to rapidly screen complex samples for a wide range of nucleic acid targets, like infectious diseases, remains unmet. Digital High-Resolution Melt (dHRM) is an emerging technology with potential to meet this need by accomplishing broad-based, rapid nucleic acid sequence identification. Here, we set out to develop a computational framework for estimating the resolving power of dHRM technology for defined sequence profiling tasks. By deriving noise models from experimentally generated dHRM datasets and applying these to in silico predicted melt curves, we enable the production of synthetic dHRM datasets that faithfully recapitulate real-world variations arising from sample and machine variables. We then use these datasets to identify the most challenging melt curve classification tasks likely to arise for a given application and test the performance of benchmark classifiers. RESULTS: This toolbox enables the in silico design and testing of broad-based dHRM screening assays and the selection of optimal classifiers. For an example application of screening common human bacterial pathogens, we show that human pathogens having the most similar sequences and melt curves are still reliably identifiable in the presence of experimental noise. Further, we find that ensemble methods outperform whole series classifiers for this task and are in some cases able to resolve melt curves with single-nucleotide resolution. AVAILABILITY AND IMPLEMENTATION: Data and code available on https://github.com/lenlan/dHRM-noise-modeling. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

How Neutrophils Meet Their End.

Neutrophil death can transpire via diverse pathways and is regulated by interactions with commensal and pathogenic microorganisms, environmental exposures, and cell age. At steady state, neutrophil turnover and replenishment are continually maintained via a delicate balance between host-mediated responses and microbial forces. Disruptions in this equilibrium directly impact neutrophil numbers in circulation, cell trafficking, antimicrobial defenses, and host well-being. How neutrophils meet their end is physiologically important and can result in different immunologic consequences. Whereas nonlytic forms of neutrophil death typically elicit anti-inflammatory responses and promote healing, pathways ending with cell membrane rupture may incite deleterious proinflammatory responses, which can exacerbate local tissue injury, lead to chronic inflammation, or precipitate autoimmunity. This review seeks to provide a contemporary analysis of mechanisms of neutrophil death.

Improving Quantitative Power in Digital PCR through Digital High-Resolution Melting.

Applying digital PCR (dPCR) technology to challenging clinical and industrial detection tasks has become more prevalent because of its capability for absolute quantification and rare target detection. However, practices learned from quantitative PCR (qPCR) that promote assay robustness and wide-ranging utility are not readily applied in dPCR. These include internal amplification controls to account for false-negative reactions and amplicon high-resolution melt (HRM) analysis to distinguish true positives from false positives. Incorporation of internal amplification controls in dPCR is challenging because of the limited fluorescence channels available on most machines, and the application of HRM analysis is hindered by the separation of heating and imaging functions on most dPCR systems. We use a custom digital HRM platform to assess the utility of HRM-based approaches for mitigation of false positives and false negatives in dPCR. We show that detection of an exogenous internal control using dHRM analysis reduces the inclusion of false-negative partitions, changing the calculated DNA concentration up to 52%. The integration of dHRM analysis enables classification of partitions that would otherwise be considered ambiguous "rain," which accounts for up to ∼3% and ∼10% of partitions in intercalating dye and hydrolysis probe dPCR, respectively. We focused on developing an internal control method that would be compatible with broad-based microbial detection in dPCR-dHRM. Our approach can be applied to a number of DNA detection methods including microbial profiling and may advance the utility of dPCR in clinical applications where accurate quantification is imperative.

Erythrocyte-Coated Nanoparticles Block Cytotoxic Effects of Group B Streptococcus ß-Hemolysin/Cytolysin.

Group B Streptococcus (GBS) emerged as a leading cause of invasive infectious disease in neonates in the 1970s, but has recently been identified as an escalating public health threat in non-pregnant adults, particularly those of advanced aged or underlying medical conditions. GBS infection can rapidly develop into life-threatening disease despite prompt administration of effective antibiotics and initiation of state-of-the-art intensive care protocols and technologies due to deleterious bacterial virulence factors, such as the GBS pore-forming toxin ß-hemolysin/cytolysin (ß-H/C). ß-H/C is known to have noxious effects on a wide range of host cells and tissues, including lung epithelial cell injury, blood brain barrier weakening, and immune cell apoptosis. Neonatal and adult survivors of GBS infection are at a high risk for substantial long-term health issues and neurologic disabilities due to perturbations in organ systems caused by bacterial- and host- mediated inflammatory stressors. Previously engineered anti-virulence inhibitors, such as monoclonal antibodies and small molecular inhibitors, generally require customized design for each different pathogenic toxin and do not target deleterious host pro-inflammatory responses that may cause organ injury, septic shock, or death. By simply wrapping donor red blood cells (RBCs) around polymeric cores, we have created biomimetic "nanosponges." Because nanoparticles retain the same repertoire of cell membrane receptors as their host cell, they offer non-specific and all-purpose toxin decoy strategies with a broad ability to sequester and neutralize various bacterial toxins and host pro-inflammatory chemokines and cytokines to attenuate the course of infectious disease. This proof-of-concept study successfully demonstrated that intervention with nanosponges reduced the hemolytic activity of live GBS and stabilized ß-H/C in a dose-dependent manner. Nanosponge treatment also decreased lung epithelial and macrophage cell death following exposure to live GBS bacteria and stabilized ß-H/C, improved neutrophil killing of GBS, and diminished GBS-induced macrophage IL-1ß production. Our results, therefore, suggest biomimetic nanosponges provide a titratable detoxification therapy that may provide a first-in-class treatment option for GBS infection by sequestering and inhibiting ß-H/C activity.

Outcomes of Infants with Mild Hypoxic Ischemic Encephalopathy Who Did Not Receive Therapeutic Hypothermia.

INTRODUCTION: Accurately diagnosing and treating infants with mild forms of hypoxic ischemic encephalopathy (HIE) is important, as the majority of neonates with signs and symptoms of HIE after birth do not meet clinical criteria for moderate or severe disease. Emerging evidence, however, suggests that infants with mild HIE (mHIE) have an increased risk for neurodevelopmental impairment (NDI). METHODS: This retrospective descriptive study examined all inborn infants ≥35 week's gestational age at a single, level III neonatal intensive care unit (NICU) in California between January 1, 2012, and December 31, 2015. International Classification of Diseases codes were used as a proxy to identify neonates with mHIE but who did not receive therapeutic hypothermia (TH). Short- and long-term neurodevelopmental outcomes were documented, including abnormal (1) brain magnetic resonance imaging within 10 days of birth suggestive of HIE, (2) electroencephalogram with electrographic seizures, (3) neurologic discharge examination, or (4) NDI following NICU discharge. RESULTS: Over the 4-year study period, 25 infants met inclusion criteria. Eight of 25 (32%) infants demonstrated neurologic impairment, defined by an abnormality in at least one of the four categories. The remaining 17 infants were without documented evidence for adverse outcomes. CONCLUSION: Our results indicate that children with mHIE are at significant risk for neurologic injury and may benefit from more aggressive interventions. Further prospective studies should be completed to determine the efficacy of TH in this specific patient population.

Emerging Technologies for Molecular Diagnosis of Sepsis.

Rapid and accurate profiling of infection-causing pathogens remains a significant challenge in modern health care. Despite advances in molecular diagnostic techniques, blood culture analysis remains the gold standard for diagnosing sepsis. However, this method is too slow and cumbersome to significantly influence the initial management of patients. The swift initiation of precise and targeted antibiotic therapies depends on the ability of a sepsis diagnostic test to capture clinically relevant organisms along with antimicrobial resistance within 1 to 3 h. The administration of appropriate, narrow-spectrum antibiotics demands that such a test be extremely sensitive with a high negative predictive value. In addition, it should utilize small sample volumes and detect polymicrobial infections and contaminants. All of this must be accomplished with a platform that is easily integrated into the clinical workflow. In this review, we outline the limitations of routine blood culture testing and discuss how emerging sepsis technologies are converging on the characteristics of the ideal sepsis diagnostic test. We include seven molecular technologies that have been validated on clinical blood specimens or mock samples using human blood. In addition, we discuss advances in machine learning technologies that use electronic medical record data to provide contextual evaluation support for clinical decision-making.

The Ontogeny of a Neutrophil: Mechanisms of Granulopoiesis and Homeostasis.

Comprising the majority of leukocytes in humans, neutrophils are the first immune cells to respond to inflammatory or infectious etiologies and are crucial participants in the proper functioning of both innate and adaptive immune responses. From their initial appearance in the liver, thymus, and spleen at around the eighth week of human gestation to their generation in large numbers in the bone marrow at the end of term gestation, the differentiation of the pluripotent hematopoietic stem cell into a mature, segmented neutrophil is a highly controlled process where the transcriptional regulators C/EBP-α and C/EBP-ε play a vital role. Recent advances in neutrophil biology have clarified the life cycle of these cells and revealed striking differences between neonatal and adult neutrophils based on fetal maturation and environmental factors. Here we detail neutrophil ontogeny, granulopoiesis, and neutrophil homeostasis and highlight important differences between neonatal and adult neutrophil populations.

Chorioamnionitis, IL-17A, and fetal origins of neurologic disease.

The Centers for Disease Control and Prevention estimate that 1 in 323 infants have cerebral palsy. Highly correlated to intrauterine infection and inflammation, the incidence of cerebral palsy has remained constant over the last few decades despite significant advances in neonatal intensive care including improved ventilator techniques, surfactant therapy, maternal steroid administration, and use of intrapartum empiric antimicrobials. Recent advances in our understanding of immune responses to infection and inflammation have identified the cytokine IL-17A as a crucial component of early proinflammatory mediators that cause brain injury associated with neurologic impairment. Remarkably, maternal inflammatory responses to in utero inflammation and infection can also lead to potentially debilitating neurologic conditions in the offspring, which often become clinically apparent during childhood and/or early adulthood. This review details the role of IL-17A in fetal and maternal proinflammatory responses that lead to fetal brain injury and neurologic sequelae, including cerebral palsy. Recent findings regarding the role of maternal inflammatory responses in the development of childhood and adult neurologic conditions, such as autism, schizophrenia, and multiple sclerosis, will also be highlighted.

IL-17 in neonatal health and disease.

Over the last few years, scientific interest in the cytokine IL-17A has intensified as its role in human health and disease has been elucidated. Discovered almost a quarter century ago, IL-17A is known to have poor biologic activity when acting alone, but attains robust actions when working synergistically with potent mediators of proinflammatory immune responses, such as IL-6 and IL-8. IL-17A is produced by specialized innate immune cells that protect host barriers from the outside world. Like sentries, these innate immune cells can "sound the alarm" through increased production of IL-17A, causing activation and recruitment of primed neutrophils and monocytes when pathogens escape initial host defenses. In this way, IL-17A promulgates mechanisms responsible for pathogen death and clearance. However, when IL-17A pathways are triggered during fetal development, due to chorioamnionitis or in utero inflammatory conditions, IL-17A can instigate and/or exacerbate fetal inflammatory responses that increase neonatal morbidities and mortality associated with common neonatal conditions such as sepsis, bronchopulmonary dysplasia (BPD), patent ductus arteriosus (PDA), and necrotizing enterocolitis (NEC). This review details the ontogeny of IL-17A in the fetus and newborn, discusses how derangements in its production can lead to pathology, and describes known and evolving therapies that may attenuate IL-17A-mediated human conditions.

FLLL32, a curcumin analog, ameliorates intestinal injury in necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease that primarily affects premature infants. It is characterized by inflammation and leukocyte infiltration that can progress to intestinal necrosis, perforation, systemic inflammatory response, and death. In this study, we examined the effect of FLLL32, a curcumin analog, on an NEC-like neonatal intestinal injury model. METHODS: NEC was induced in CD-1 mice pups using the Paneth cell ablation and Klebsiella infection model. Pups were divided into sham, NEC, and NEC + FLLL32 groups. At the end of the experiment, pups were euthanized; whole blood and small intestines were harvested. Intestinal inflammatory cytokine profile, in vivo intestinal permeability using serum fluorescein isothiocyanate-dextran, and histological injury scores were compared between the groups. RESULTS AND CONCLUSION: FLLL32-treated pups had lower intestinal injury, improved intestinal permeability, and lower proinflammatory cytokine profiles compared to those in untreated pups with NEC. These results suggest that FLLL32 plays a protective role in NEC.

Alterations in neonatal neutrophil function attributable to increased immature forms.

At birth neonatal neutrophil composition differs from that of adults due to a higher number of circulating immature forms. To date only a single study has evaluated neutrophil performance based on cell maturity. For this study, we examined functional differences in chemotaxis and phagocytosis between neonatal and adult neutrophils based on cell development and labor exposure. METHODS: Neutrophils were obtained by venipuncture from adults and cord blood from healthy term neonates delivered vaginally or by cesarean section. Transwells and the chemoattractant fMLP were used to evaluate chemotaxis. Phagocytosis assays were performed using GFP-labeled E.coli (RS218) and whole blood. Neutrophil maturation was measured by an accurate and verified flow cytometry technique using the markers CD45, CD11b, and CD16. QuantiGene Plex and Procarta immunoassays were used to determine cytokine and chemokine gene expression and protein concentration, respectively. RESULTS: Labor exposure did not alter neonatal neutrophil function in this study. Neonatal and adult mature neutrophils performed chemotaxis and phagocytosis equally well, while immature forms showed marked impairments. Neonatal immature granulocytes, though, completed chemotaxis more proficiently than those of adults. Although cytokine and chemokine levels varied between neonatal and adult groups, no differences were detected in neonates based upon labor exposure. CONCLUSION: Historically documented functional impairments of neonatal neutrophils may be due to the increased number of developmentally immature forms at birth rather than absolute global deficiencies.