Giulio Seganti

CpG Drives Human Transitional B Cells to Terminal Differentiation and Production of Natural Antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.

Procalcitonin in detecting neonatal nosocomial sepsis

Objective To investigate the accuracy of procalcitonin (PCT) as a diagnostic marker of nosocomial sepsis (NS) and define the most accurate cut-off to distinguish infected from uninfected neonates. Setting Six neonatal intensive care units (NICUs). Patients 762 neonates admitted to six NICUs during a 28-month observational study for whom at least one serum sample was taken on admission. Main outcome measures Positive and negative predictive values at different PCT cut-off levels. Results The overall probability of an NS was doubled or more if PCT was >0.5 ng/ml. In very-low-birth-weight (VLBW) infants, a cut-off of >2.4 ng/ml gave a positive predictive value of NS near to 50% with a probability of a false-positive diagnosis of NS in about 10% of the patients. Conclusions In VLBW neonates, a serum PCT value >2.4 ng/ml prompts early empirical antibiotic therapy, while in normal-birth-weight infants, a PCT value ≤2.4 ng/ml carries a low risk of missing an NS.

Determinants of Nosocomial Infection in 6 Neonatal Intensive Care Units: An Italian Multicenter Prospective Cohort Study

BACKGROUND Nosocomial infections are still a major cause of morbidity and mortality among neonates admitted to neonatal intensive care units (NICUs). OBJECTIVE To describe the epidemiology of nosocomial infections in NICUs and to assess the risk of nosocomial infection related to the therapeutic procedures performed and to the clinical characteristics of the neonates at birth and at admission to the NICU, taking into account the time between the exposure and the onset of infection. DESIGN A multicenter, prospective cohort study. PATIENTS AND SETTING A total of 1,692 neonates admitted to 6 NICUs in Italy were observed and monitored for the development of nosocomial infection during their hospital stay. METHODS Data were collected on the clinical characteristics of the neonates admitted to the NICUs, their therapeutic interventions and treatments, their infections, and their mortality rate. The cumulative probability of having at least 1 infection and the cumulative probability of having at least 1 infection or dying were estimated. The hazard ratio (HR) for the first infection and the HR for the first infection or death were also estimated. RESULTS A total of 255 episodes of nosocomial infection were diagnosed in 217 neonates, yielding an incidence density of 6.9 episodes per 1,000 patient-days. The risk factors related to nosocomial infection in very-low-birth-weight neonates were receipt of continuous positive airway pressure (HR, 3.8 [95% confidence interval {CI}, 1.7-8.1]), a Clinical Risk Index for Babies score of 4 or greater (HR, 2.2 [95% CI, 1.4-3.4]), and a gestational age of less than 28 weeks (HR, 2.1 [95% CI, 1.2-3.8]). Among heavier neonates, the risk factors for nosocomial infection were receipt of parenteral nutrition (HR, 8.1 [95% CI, 3.2-20.5]) and presence of malformations (HR, 2.3 [95% CI, 1.5-3.5]). CONCLUSIONS Patterns of risk factors for nosocomial infection differ between very-low-birth-weight neonates and heavier neonates. Therapeutic procedures appear to be strong determinants of nosocomial infection in both groups of neonates, after controlling for clinical characteristics.

Role of mannose-binding lectin in nosocomial sepsis in critically ill neonates.

We investigated the association of mannose-binding lectin (MBL) serum levels with nosocomial sepsis (NS), their changes overtime during infection, their relation with pathogens, with the MBL2 genotype and their relationship with mortality. In a prospective observational study, we included 365 critically ill neonates: 261 had no infection and 104 had at least 1 septic event. The median MBL serum concentration was significantly lower in infected than in noninfected neonates (p < 0.001). Low MBL levels on admission increased the risk of infection, independently from gestational age and invasive procedures. The median peak MBL level during infection was higher than the median level on admission (p < 0.001) and was correlated with it (r(2) = 0.83, p < 0.001). Moreover, MBL levels on admission were not associated with death (OR = 0.80, 95% CI = 0.56-1.14, p = 0.21). Similarly, no association was found between MBL peak levels during infection and death among infected neonates (OR = 1.10, 95% CI = 0.78-1.57, p = 0.57). In 127 neonates (42 infected) genotyped for exon-1 and -221 promoter MBL2 variants, we did not find significant difference in the frequencies of MBL2 genotypes between infected and noninfected neonates. Moreover, no association was found between MBL2 genotypes and death.

A polymorphism in the macrophage migration inhibitory factor promoter is associated with bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common adverse outcome of prematurity, causing severe morbidity and mortality. The cytokine macrophage migration inhibitory factor (MIF) has been recently shown to favor murine fetal lung development. In this prospective study, we evaluate the expression of MIF in the lung and in the serum of preterm infants (n = 50) and investigate whether the −173 G/C MIF promoter polymorphism is associated with the risk of BPD (n = 103). MIF was highly expressed in lung tissue from preterm infants. Serum MIF levels were measured by ELISA at d 1 after birth. MIF levels were increased [median (interquartile range), 71.01 (44.9–162.3) ng/mL], particularly in those infants with RDS [110.4 (59.4–239.2) ng/mL] compared with healthy adults [2.4 (1.2–5.0) ng/mL], (p < 0.001). The MIF −173*C allele, which predisposes to higher MIF production, was associated with a lower incidence of BPD (OR, 0.2; 95% CI, 0.04–0.93), independently from mechanical ventilation and oxygen exposure (p = 0.03). In conclusion, these data show that MIF expression is increased in lung and serum of preterm infants and suggest that the high producing MIF −173*C allele may be a protective factor for BPD.

Congenital Varicella Syndrome: Still a Problem?

A woman contracted chickenpox in the 12th week of gestation. Her general practitioner and later the consultant obstetrician warned her about the small risk of giving birth to a disabled child. She decided to continue the pregnancy without undergoing invasive tests to diagnose fetal intrauterine infection. Symptoms of congenital varicella syndrome (CVS) were detected by ultrasound in the 29th and 34th weeks of gestation. On admission to hospital, the baby was not considered infectious and was not isolated because polymerase chain reaction analysis to detect varicella zoster virus (VZV) DNA in the blood, cerebrospinal fluid, saliva, skin scrapings and feces gave negative results. He was also not separated from his mother. The mother was without clinical complications. Varicella during pregnancy may result in VZV transmission to the fetus or newborn. Intrauterine VZV infection in the first 28 weeks of gestation may result in CVS with limb deformities, brain abnormalities and mental retardation. Usually the newborn is not infectious, and therapy and isolation are unnecessary. When the mother catches the infection in the second trimester, the newborn may manifest shingles in the first 2 years of life. A maternal rash erupting 5 days before to 2 days after delivery is frequently associated with clinically severe varicella in the newborn, leading to high mortality if untreated. Then the newborn is infectious and must be isolated. This case report underlines the need for expert medical counseling for women who contract chickenpox at any time during pregnancy. It also underlines the importance of immunizing susceptible women of childbearing age before they become pregnant.

Birth of a child with congenital heart disease: emotional reactions of mothers and fathers according to time of diagnosis

Abstract Objective: To evaluate emotional distress, depression and quality of life in parents of infants with severe congenital heart disease (CHD) during their first hospitalization. Methods: A pilot study for 38 parental couples of infants with CHD hospitalized within the 3 months of life. Parents filled up three self-administered questionnaires. We compared differences in the variables measuring emotional distress, depression and quality of life between mothers and fathers, and between prenatal and postnatal diagnosis. Results: Stress and depression levels were significantly higher in mothers than in fathers (stress: 81.8% mothers versus 60.6% fathers; depression: 45.7% mothers versus 20.0% fathers). No difference were found between prenatal and postnatal groups in any field tested but, in percentage, mothers receiving prenatal diagnosis were more depressed, whereas those receiving postnatal diagnosis were more stressed. Fathers showed same tendency. Conclusions: Parents of newborns with severe CHD, especially mothers, need psychological support during their child’s hospitalization. Parents of children diagnosed prenatally may need counseling throughout pregnancy to help them recover from the loss of the imagined healthy child.

Cocaine abuse in pregnancy: Its evaluation through hair analysis of pathological new-borns

Cocaine abuse in pregnancy has been evaluated through toxicological analysis of hair from 123 pathological new-borns admitted in an intensive care division. The new-borns were affected with malformations, low gestational age, low birth weight, respiratory distress. A control group of 39 healthy new-borns was also analysed. Hair samples (about 50 mg) were enzymatically digested and directly analysed by RIA. Samples positive to the preliminary screening were extracted by SPE columns and injected in GC/MS. Results obtained showed 3 positive samples (2.4%). All cocaine babies had low gestational age, low birth weight, two of them showed heart malformation, one kidney malformation and one genital malformation. None of the control new-born resulted positive for cocaine. These results show a worrying trend of cocaine diffusion in the Italian population.

Isolated fetal ascites due to Budd-Chiari syndrome.

A 23-year-old woman, gravida 2, para 1, presented at 33 weeks’ gestation with isolated fetal ascites without congenital defects or associated skin edema. The previous pregnancy had been uneventful with a healthy baby delivered by Cesarean section. In the index pregnancy, fetal biparietal diameter and femur length measured on ultrasound examination were consistent with gestational age. The mother had a bicornuate uterus and a single kidney. Serological testing during the first trimester of pregnancy and at 33 weeks’ gestation was negative for cytomegalovirus, Epstein–Barr virus, rubella virus, Toxoplasma gondii, and herpes simplex virus Types 1 and 2. Fetal karyotyping following amniocentesis revealed 46,XY. At 35 weeks’ gestation, a 2700-g male was delivered by repeat Cesarean section. Ultrasound imaging showed moderate hepatomegaly and mild ascites. Liver function tests were normal and serological analysis for hepatitis was negative. At 29 days after birth surgery for an inguinal hernia was carried out. The postoperative course was complicated by generalized edema and evident abdominal distension due to the increasing hepatomegaly (Figure 1). Polymerase chain reaction analysis of urine, saliva and blood excluded cytomegalovirus infection. Laboratory studies for the diagnosis of metabolic diseases, including galactosemia, glycogenosis Type 1 or 3, α1antitrypsin deficiency, tyrosinemia Type 1, neonatal hemochromatosis and Niemann–Pick disease Type C, were normal. An abdominal computed tomographic scan at 48 postnatal days showed enlargement of the liver and spleen, without parenchymal lesions but with hepatic venous obstruction. The celiac trunk, hepatic artery and splenic artery were filled with contrast medium, but the portal vein was not completely visualized and a distinct collateral circulation of the azygos vein was present, leading to a diagnosis of Budd–Chiari syndrome. Investigation for familial thrombophilia showed that the baby was heterozygous for the factor V Leiden mutation. Budd–Chiari syndrome is a rare disorder, resulting from occlusion of hepatic venous drainage by hepatic vein thrombosis. The acute syndrome is characterized by sudden onset of ascites, causing abdominal distension, hepatomegaly, jaundice and hepatic failure. In the chronic form, the disease progresses slowly and symptoms develop over months or even years. Budd–Chiari syndrome associated with familial thrombophilia is extremely rare in newborn babies1 and has not been reported previously as a cause of fetal ascites. Fetal ascites is easily diagnosed by ultrasound imaging, where it appears as fluid in the peritoneal cavity. In our case, fetal ascites was an isolated Figure 1 Radiograph of the neonatal abdomen showing enlargement of the liver and spleen with displacement of the bowel in the lower abdominal quadrant.

15 Determinants of Nosocomial Infection (NI) in Six Italian Neonatal Intensive Care Units (NICUs).

As care improves many neonates with life-threatening disorders now survive. Nonetheless Nosocomial Infections (NI) are still a major cause of morbidity and mortality in NICUs. A prospective multicentric surveillance study was conducted in six Italian NICUs to describe the epidemiologic profile and determinants of NI in NICU. 1692 neonates, consecutively admitted to the NICUs from July 2000 to October 2002 and monitored for the development of NI were enrolled into the study. The standard definition criteria for NI formulated by the Centers for Disease Control in Atlanta were used. The cumulative probability and hazard ratios (HR) for the first episode of infection were estimated by the Kaplan-Meier method and the Cox model. A total of 217 neonates had 255 episodes of NI. The incidence rate of NI was 7 per 1000 patient-days. The cumulative probability of first infection was 20% (95% CI, 7.50–23.30) and 27.6% (95 CI, 23.20–32.80) at 30 and 60 days after admission to the NICU. After adjustment for the severity of illness, the main risk factors related to NI in very-low-birth-weight neonates (VLBW) were surgical procedures (HR 2.69;95% CI 0.60–12.08), nasal ventilation (CPAP) (HR, 2.51; 95% CI, 0.93–6.76), continuous enteral feeding (HR 1.89; 95% CI, 0.20–17.50), mechanical ventilation (HR, 1.70; 95%CI, 0.72–4.00) and intravenous infusions (HR 1.46; 95% CI, 0.32–6.52). Among neonates with a birth weight over 1500 g, risk factors for NI were parenteral nutrition with lipid emulsion (HR, 12.41; 95% CI, 4.19–36.78), surgical procedures (HR 2.78; 95% CI, 0.82–9.44), and intravenous infusions (HR, 2.63; 95% CI, 0.27–25.53). Risk factors for NI were related more to the severity of illness than to healthcare procedures in VLBW babies and more to medications among neonates weighing more than 1500 g at birth.Supported by a Grant from the Italian Ministry of Health, N 99010661