Identifying immune signatures of sepsis to increase diagnostic accuracy in very preterm babies.

Bacterial infections are a major cause of mortality in preterm babies, yet our understanding of early-life disease-associated immune dysregulation remains limited. Here, we combine multi-parameter flow cytometry, single-cell RNA sequencing and plasma analysis to longitudinally profile blood from very preterm babies (<32 weeks gestation) across episodes of invasive bacterial infection (sepsis). We identify a dynamically changing blood immune signature of sepsis, including lymphopenia, reduced dendritic cell frequencies and myeloid cell HLA-DR expression, which characterizes sepsis even when the common clinical marker of inflammation, C-reactive protein, is not elevated. Furthermore, single-cell RNA sequencing identifies upregulation of amphiregulin in leukocyte populations during sepsis, which we validate as a plasma analyte that correlates with clinical signs of disease, even when C-reactive protein is normal. This study provides insights into immune pathways associated with early-life sepsis and identifies immune analytes as potential diagnostic adjuncts to standard tests to guide targeted antibiotic prescribing.

Intravenous antibiotics in preterm infants have a negative effect upon microbiome development throughout preterm life.

BACKGROUND: Intestinal dysbiosis is implicated in the origins of necrotising enterocolitis and late-onset sepsis in preterm babies. However, the effect of modulators of bacterial growth (e.g. antibiotics) upon the developing microbiome is not well-characterised. In this prospectively-recruited, retrospectively-classified, case-control study, high-throughput 16S rRNA gene sequencing was combined with contemporaneous clinical data collection, to assess the within-subject relationship between antibiotic administration and microbiome development, in comparison to preterm infants with minimal antibiotic exposure. RESULTS: During courses of antibiotics, diversity progression fell in comparison to that seen outside periods of antibiotic use (-0.71units/week vs. + 0.63units/week, p < 0.01); Enterobacteriaceae relative abundance progression conversely rose (+ 10.6%/week vs. -8.9%/week, p < 0.01). After antibiotic cessation, diversity progression remained suppressed (+ 0.2units/week, p = 0.02). CONCLUSIONS: Antibiotic use has an acute and longer-lasting impact on the developing preterm intestinal microbiome. This has clinical implications with regard to the contribution of antibiotic use to evolving dysbiosis, and affects the interpretation of existing microbiome studies where this effect modulator is rarely accounted for.

An investigation into the reporting and management of late terminations of pregnancy (between 22 +0 and 26 +6 weeks of gestation) within NHS Hospitals in England in 2006: the EPICure preterm cohort study.

OBJECTIVE: To review all late terminations of pregnancy, between 22(+0) and 26(+6) weeks of gestation, collected as part of the EPICure2 study. DESIGN: Prospective cohort study. SETTING: All National Health Service (NHS) hospitals providing perinatal services for extremely preterm infants. POPULATION: All births between 22(+0) and 26(+6) weeks of gestation in England during 2006. METHODS: Data were collected for the defined cohort of births, including terminations of pregnancy, by designated unit staff using a standardised questionnaire. Rigorous validation processes were established to ensure comprehensive data collection. Gestational age was validated using a hierarchical classification of scan dates, certain date of last menstrual period and working gestation. Data for terminations of pregnancy (TOPs) were categorised into two groups, terminations for fetal abnormality and for maternal or fetal compromise, and were analysed in terms of their reporting, management and outcomes. MAIN OUTCOME MEASURES: Classification, rate of feticide and outcome following TOP. RESULTS: Of 3782 births between 22(+0) and 26(+6) weeks of gestation, 647 (17.1%) were TOPs; of these 584 (90.3%) were for fetal abnormality and 63 (9.7%) for maternal or fetal compromise. Feticide was carried out in 489 of 584 (83.7%) TOPs for fetal abnormality, and in 38 of 63 (60.3%) of the TOPs for maternal or fetal compromise. Live births resulted following 2.2% TOPs for fetal abnormality and 4.8% TOPs for maternal or fetal compromise. CONCLUSION: Terminations of pregnancy represent a relatively large proportion of very preterm births. Fetal abnormalities are the main cause for these terminations, and most include feticide. Better screening strategies are required to avoid the need for late terminations of pregnancy for fetal abnormalities.

Perinatal inflammation influences but does not arrest rapid immune development in preterm babies.

Infection and infection-related complications are important causes of death and morbidity following preterm birth. Despite this risk, there is limited understanding of the development of the immune system in those born prematurely, and of how this development is influenced by perinatal factors. Here we prospectively and longitudinally follow a cohort of babies born before 32 weeks of gestation. We demonstrate that preterm babies, including those born extremely prematurely (<28 weeks), are capable of rapidly acquiring some adult levels of immune functionality, in which immune maturation occurs independently of the developing heterogeneous microbiome. By contrast, we observe a reduced percentage of CXCL8-producing T cells, but comparable levels of TNF-producing T cells, from babies exposed to in utero or postnatal infection, which precedes an unstable post-natal clinical course. These data show that rapid immune development is possible in preterm babies, but distinct identifiable differences in functionality may predict subsequent infection mediated outcomes.

Association of prematurity, lung disease and body size with lung volume and ventilation inhomogeneity in unsedated neonates: a multicentre study.

BACKGROUND: Previous studies have suggested that preterm birth with or without subsequent chronic lung disease is associated with reduced functional residual capacity (FRC) and increased ventilation inhomogeneity in the neonatal period. We aimed to establish whether such findings are associated with the degree of prematurity, neonatal respiratory illness and disproportionate somatic growth. METHODS: Multiple breath washout measurements using an ultrasonic flowmeter were obtained from 219 infants on 306 test occasions during the first few months of life, at three neonatal units in the UK and Australia. Tests were performed during unsedated sleep in clinically stable infants (assigned to four exclusive diagnostic categories: term controls, preterm controls, respiratory distress syndrome and chronic lung disease). The determinants of neonatal lung function were assessed using multivariable, multilevel modelling. RESULTS: After adjustment for age and body proportions, the factors gestation, intrauterine growth restriction and days of supplemental oxygen were all significantly associated with a reduced FRC. In contrast, increased ventilation inhomogeneity (elevated lung clearance index) was only significantly associated with duration of supplemental oxygen. After adjusting for continuous variables, diagnostic category made no further contribution to the models. Despite using identical techniques, unexpected inter-centre differences occurred, associated with the equipment used; these did not alter the negative association of preterm delivery and disease severity with lung function outcomes. CONCLUSION: Reduction in FRC is independently associated with prematurity, intrauterine growth restriction and severity of neonatal lung disease. Determinants of lung function shortly after birth are highly complex in different disease groups.

Measurement of pulmonary capillary blood flow in infants by plethysmography.

An accurate method for measuring effective pulmonary capillary blood flow (Qc eff) in infants has been developed with an adaptation of the plethysmographic technique. Measurements were made on 19 preterm. 14 small-for-dates, and 7 fullterm normal infants with a constant volume whole body plethysmograph in which the infant rebreathed nitrous oxide. There was a highly significant correlation between Qc eff and body weight, and this relationship was unaffected by premature delivery or intrauterine growth retardation. Mean Qc eff in preterm, small-for dates, and fullterm infants was 203, 208 and 197 ml min-1 kg-1, respectively, with no significant differences between the groups. A significant negative correlation existed between Qc eff and haematocrit in the preterm infants. There was no relationship between weight standardized Qc eff and postnatal age in any of the groups. With this technique, it was possible to readily recognise the presence of rapid recirculation (indicative of shunting) in several of the infants, suggesting that rebreathing methods for the assessment of Qc eff should not be applied indiscriminately during the neonatal period. By taking care to overcome the potential sources of technical error, it was possible to obtain highly reproducible results of Qc eff in infants over a wider age range than has been previously reported.

The EPICure study: associations and antecedents of neurological and developmental disability at 30 months of age following extremely preterm birth.

AIMS: To describe perinatal factors associated with later morbidity among extremely preterm children at 30 months of age corrected for prematurity. POPULATION: Of 308 surviving children born at

Are deficits of arachidonic and docosahexaenoic acids responsible for the neural and vascular complications of preterm babies?

We review evidence suggesting that pre- or postnatal deficits of arachidonic acid (AA) and docosahexaenoic acid (DHA) together with underdeveloped antioxidant protection contribute to neurovisual developmental disorders and other complications of premature birth. These two synergistic deficits occur at a time when 70% of energy is focused on brain development and when the brain and blood vessels are growing at high speed. The types of essential fatty acids fed to preterm babies bear no relation to what the infant would have received had it remained a fetus. This failure to meet essential fatty acid requirements exacerbates the AA and DHA deficits seen at birth; furthermore, the immature superoxide defenses remain depressed until the expected date of delivery. Deficits of these systems, which are required for cell membranes, the endothelium, and neural tissue, could provide the biochemical prerequisite for the membrane disorders to which these babies are at high risk: intraventricular hemorrhage, periventricular leucomalacia, retinopathy of prematurity, and bronchopulmonary dysplasia. Although poor vascular development during fetal and neonatal life may be repaired, the structural and antioxidant deficits identified in preterm babies may impair blood vessel development with long-term consequences. The conclusion drawn from this review is that present parenteral and enteral lipid nutrition for preterm babies is flawed and could be pathogenic. Full-term milk composition is the basis for the design of preterm infant foods, but full-term milk is different from the placental product that is rich in AA and DHA. Preterm lipid nutrition should be revised to be more in line with placental lipid transfer to the fetus.

Blood resistivity and its implications for the calculation of cardiac output by the thoracic electrical impedance technique.

A relationship has been established between the haematocrit and the resistivity of whole blood at 37 degrees C for neonates and adults not suffering from renal failure. Values of resistivity obtained in this way were substituted into Kubicek's equation for stroke volume by the electrical impedance technique, the signal pick-up electrodes being placed in standard positions. The calculated cardiac outputs were then compared with those obtained simultaneously from other techniques such as the dye dilution method for adults and the measurement of pulmonary effective capillary blood flow by rebreathing nitrous oxide in neonates. It was found that the impedance method overrestimates under these circumstances, and other workers have shown that this is likely to be due to a contribution from the right heart. A haemotocrit-dependent correction factor has been employed in the case of neonates to align the impedance and nitrous oxide results.

Persistence of the Hering-Breuer reflex beyond the neonatal period.

There is conflicting evidence regarding the persistence of the Hering-Breuer reflex (HBR) beyond the 1st wk of life. This study was designed to assess the influence of postnatal age on the HBR. The airway occlusion technique was used to assess changes in respiratory timing during stimulation of the HBR in healthy full-term unsedated infants measured shortly after birth and at 6-8 wk of life. The strength of the HBR was assessed from the relative change in expiratory time (TE) after end-inspiratory occlusion compared with resting TE during spontaneous breathing. Paired studies were performed in 31 infants at approximately 2 days and 6 wk of age. There was a significant increase in TE during each occlusion in every infant irrespective of age at measurement. No maturational changes were observed. The increase in TE after end-inspiratory occlusion was 91.9 +/- 31.6% (SD) (range 38-158%) at approximately 2 days and 89.8 +/- 30.7% (range 44-175%) at approximately 6 wk. We conclude that the activity of the HBR during tidal breathing persists beyond the neonatal period and that there is no statistically significant change in its strength during the first 2 mo life in healthy infants during natural sleep.

Effect of periodic thermal stimulus on heart rate in term newborn infant.

We studied heart rate changes in 25 term infants aged 1-7 days in quiet sleep during periodic thermal stimulation of one foot to widen the range of frequencies previously studied by others and to develop spectral analysis methods to quantify responses to thermal and other periodic sensory stimuli. The stimulation frequency was 0.10 Hz in all babies and ranged from 0.05 to 0.15 Hz in some. At 0.10 Hz, there was 1) an increase in spectral power at the frequency of stimulation (P < 0.001), 2) a tendency for overall low-frequency power to increase, 3) a reduction in respiratory sinus arrhythmia (P < 0.025), and 4) attenuation in the response between the first and second minute of stimulation (P < 0.01). At other frequencies of stimulation, essentially similar results were obtained. Respiration and other types of periodic sensory stimulation may also entrain the heart rate; we raise the question of whether low-frequency oscillations in heart rate are in fact related to thermoregulation or are a nonspecific feature of integrative processes in the brain stem.

Prevention of neural tube defects in an urban health district.

An evaluation of voluntary prenatal screening for neural tube defects with serum alphafetoprotein (s-AFP) is presented. During a three year period, there were 52 fetuses with neural lesions. Of 46 mothers who bore a fetus with an open neural lesion, 25 were detected, of whom 23 agreed to the termination of their pregnancies. An unscreened group of 2331 mothers (17% of all deliveries) produced 14 fetuses with neural tube defects, an incidence of 6.0 per 1000, 1.8 times the incidence (3.3 per 1000) in the screened group. Pitfalls occurring in the assessment of hyper-alphafetoproteinemia included inaccurate gestational dating and allowance for excessive body weight. The results of a robust AFP-NTD screening programme supported in selected cases by ultrasonar visualisation of the spine argue for its continuance in this district.

Assessment of passive respiratory compliance in healthy preterm infants: a critical evaluation.

The airway occlusion techniques for assessing passive respiratory mechanics have become well established methods in fullterm neonates and older infants. The single breath technique (SBT) is frequently used for assessing lung function in intubated infants on neonatal intensive care units. However, less is known about the reliability of these quick and noninvasive techniques in healthy preterm infants. The aim of this study was to evaluate these methods in healthy unintubated preterm infants to facilitate both establishment of reference values and more meaningful interpretation of lung function assessments in the neonatal unit. Forty-seven studies were attempted in 31 healthy preterm infants (gestational age 29-36 weeks; body weight 1.88 +/- 0.28 kg; mean +/- SD) during the first 2 weeks of life, using both the multiple occlusion technique (MOT) and the SBT. Whereas technically acceptable respiratory system compliance (Crs) data from either the MOT or the SBT were obtained on 37 occasions in 25 infants, satisfactory results from both techniques were achieved only on 22 occasions. In these infants mean +/- SD Crs was 28.1 +/- 5.2 mL kPa-1 when assessed by MOT and 29.1 +/- 6.0 mL kPa-1 when using the SBT. The mean difference between technically satisfactory paired Crs values obtained with MOT and SBT was less than 5% (range, +28 to -18%). By contrast, in infants in whom data were invalidated as a result of expiratory airflow braking, failure to relax or instability of the end-expiratory level, gross discrepancies occurred between the techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

A new microtransducer catheter for measuring esophageal pressure in infants.

Measurement of esophageal pressure, as a reflection of pleural pressure, is essential for assessment of dynamic lung mechanics in neonates and infants. Conventionally, an esophageal balloon or a fluid-filled catheter is used, but considerable skill is required to obtain accurate results. Both devices have problems, and failure to achieve valid occlusion tests have been reported, particularly in small infants with lung disease. Recently, a flexible #3 French gauge (FG) microtransducer catheter (MTC, Dräger Netherlands) has become available for medical monitoring. We have assessed the accuracy and feasibility of using this device for measuring lung mechanics in 51 spontaneously breathing infants and small children aged 1 day to 24 months (weight 1.35 to 12.0 kg), 9 of whom were healthy neonates, the remainder suffering from a variety of cardio-respiratory diseases, and in 18 sick ventilated infants (weight 0.6 to 4.0 kg). Positioning of the catheter was well tolerated by all infants. The ratio of esophageal to airway opening pressure changes (delta Pes:delta Pao) ranged from 0.94 to 1.09 [mean (SD) 1.013 (0.03)] for the spontaneously breathing infants and from 0.98 to 1.06 [mean (SD) 1.003 (0.02)] In the ventilated infants with no significant difference in this ratio between the two groups (p = 0.16). This new generation of catheter tip pressure transducers may provide a simpler and more reliable tool for assessing transpulmonary pressure changes in infants than has previously been available.

Influence of sedation on the Hering-Breuer inflation reflex in healthy infants.

The airway occlusion technique for measuring passive respiratory mechanics in infants relies on an ability to evoke the Hering-Breuer inflation reflex (HBR). However, the persistence of this reflex beyond the early newborn period remains controversial. We have recently demonstrated that there is no change in the strength of this reflex during the first two months of life in healthy infants during natural sleep. Measurements beyond this immediate newborn period are difficult without sedation, but it is unclear whether sedation itself may influence this reflex. To investigate the influence of sedation, the HBR was measured in 66 healthy, full-term infants aged 4-8 weeks. Thirty-three infants were measured during natural sleep, and 33 after triclofos sodium sedation (75 mg.kg-1). The strength of the HBR was assessed from the change in expiratory time (TE) following brief end-inspiratory airway occlusion, as compared to TE during spontaneous breathing. The mean increase in TE following occlusion was 89.45% (SD, 29.8; range, 44-175) in infants sleeping naturally, and 92.42% (SD, 31.2; range, 34-179) in sedated infants. Using unpaired t tests, no statistically significant difference was found between groups (P = 0.7516). We conclude that the strength of the HBR in healthy infants is not influenced by sedation with triclofos sodium, in doses normally used for lung function testing.

Arachidonic and docosahexaenoic acids are strongly associated in maternal and neonatal blood.

BACKGROUND: The red cell membrane fatty acid composition has frequently been used as an index of essential fatty acid (EFA) nutrition. After birth there is a decline in plasma arachidonic acid (AA) and docosahexaenoic (DHA) acids in babies fed on conventional formula which contains only the parent linoleic and alpha-linolenic acids. In human studies, the red cell phosphoglyceride composition appears to be more constant than that of plasma. In infants fed fish oil without AA, the AA proportions fall in the plasma but much less so in the red cells. This result might be considered to mean that there is no need for preformed AA. On the other hand, in a study where the levels of AA fell there was reduction of infant growth. Indeed, where cell membrane composition does change there is often an associated alteration in physiological functions of membranes. We therefore felt it worth investigating the balance between AA and DHA in a physiological situation where plasma levels are known to change, namely in pregnancy. PURPOSE: The aim of the study was to investigate a relationship between blood phosphoglyceride AA and DHA in pregnant women and neonates. SUBJECTS: Health pregnant women from London, England (n=193) and their term babies (n=45); healthy pregnant women from Seoul, South Korea (n=40) and their term babies (n=40); and preterm neonates (n=72) from London. METHOD: Blood samples were taken from British and Korean pregnant women during the third trimester, and from term and preterm babies at birth. These samples were taken for routine monitoring purposes in Korea and were a part of a study on pregnancy outcome for which ethical permission was granted from the East London and The City Health Authority and Lambeth, Southwark and Lewisham Health Authority. Approval was also obtained from the Ethical Committee of the Asan Medical Centre, Seoul, South Korea. RESULTS: AA and DHA correlated in plasma choline phosphoglycerides (CPG) of the British mothers (r=0.52 P<0.0001). The correlation coefficients and significance were much stronger in the red cell CPG and even more so in the term and preterm infant red cell CPGs ( r=0.75, 0.80 and 0.88, respectively). Similarly, AA and DHA correlated in red cell CPGs of the Korean women and their term babies. There was also a significant relationship between the two fatty acids in red cell ethanolamine phosphoglycerides in the mothers and their babies. Both linoleic (LA) and alpha-linolenic acids (ALA) were inversely associated with AA and DHA in some of the phosphoglyceride fractions of the mothers and babies. CONCLUSIONS: Although AA and DHA have different primary dietary origins, there were significant relationships between AA and DHA in the phosphoglycerides of the red cell membrane. This finding seems surprising if the red cell composition is determined by diet. These results suggest a physiological mechanism which attempts to maintain an appropriate balance between AA and DHA. It is plausible that there is an optimum balance between AA and DHA for membrane stability, deformability, enzyme and receptor function. SPONSORSHIP: The British Diabetic Association, March of Dimes Birth Defects Foundation and The Christopher H.R. Reeves Charitable Trust. European Journal of Clinical Nutrition (2000) 54, 50-56

Probiotics for preterm infants?

Infants nursed in special care baby units develop an abnormal pattern of microbial colonisation, which may contribute to disease. Enteric feeding of live microbial supplements (probiotics) may provide benefit to such infants and help to prevent diseases such as neonatal necrotising enterocolitis.

Decision making and modes of death in a tertiary neonatal unit.

AIMS: To study the frequency and reason for withdrawal/withholding of life sustaining treatment (LST) and do not resuscitate (DNR) orders in infants who died in a tertiary neonatal unit. METHODS: Infants who died at Homerton University Hospital between January 1998 and September 2001 were studied by retrospective analysis of patient records. RESULTS: The case notes of 71 (84%) of 85 infants who died were studied. Mode of death was withdrawal of LST in 28 (40%), DNR in 11 (15%), withholding of LST in two (3%), and natural in 30 (42%) infants. Withdrawal of LST was discussed with the parents of 39 seriously ill infants; 28 (72%) parents agreed. There was no difference in birth weight and gestational age of babies whose parents agreed or refused withdrawal of LST. White and Afro-Caribbean parents and those from the Indian subcontinent (20 of 23) were more likely to agree to withdrawal of LST than Black African or Jewish (eight of 16, p = 0.015) parents. The median age at withdrawal of LST was 4 days (range 1-57). The median duration between discussion and the parents agreeing to withdrawal of LST was 165 minutes (range 30-2160), and median duration between withdrawal of LST and death was 22 minutes (range 5-210). The most common reason for withdrawal of LST was complications of extreme prematurity (68%). CONCLUSION: The most common mode of death was withdrawal of LST, and the most common reason was complications of extreme prematurity. The ethnic and cultural background of the parents influenced agreement to withdrawal of LST.

The EPICure study: growth and associated problems in children born at 25 weeks of gestational age or less.

AIM: To define growth outcomes of a geographically defined population of extremely preterm babies. POPULATION: The EPICure study identified all surviving children in the United Kingdom and Ireland born at < or = 25 weeks 6 days gestation between March and December 1995. Of 308 survivors, 283 (92%) were evaluated at 30 months of age corrected for prematurity. METHODS: Growth was measured as part of a medical and full neurodevelopmental assessment. Growth parameters were evaluated in relation to other 30 month outcomes and perinatal variables. RESULTS: The children were smaller in each of the five growth measures compared with published population norms: mean (SD) standard deviation scores were -1.19 (1.32) for weight, -1.40 (1.37) for head circumference, -0.70 (1.19) for height, -1.00 (1.38) for body mass index, and -0.75 (0.95) for mid-upper arm circumference. Despite being of average size at birth, children were significantly lighter with smaller head circumferences at the expected date of delivery, compared with population norms, and only weight showed later catch up, by 0.5 SD. Poorer growth was found in children whose parents reported feeding problems and with longer duration of oxygen dependency, as a marker for neonatal respiratory illness. Although severe motor disability was associated with smaller head circumference, overall there was no relation between Bayley scores and head growth. CONCLUSIONS: Poor growth in early childhood is common in extremely preterm children, particularly when prolonged courses of systemic steroids have been given for chronic lung disease. Improving early growth must be a priority for clinical care.

Trisomy 22 and intersex.

Complete trisomy 22, with or without mosaicism, has been reported as a distinct syndrome. In this report an infant is described who was externally male but with female rudimentary internal organs and whose karyotype was 47,XX+22.