Survey of neonatal candidiasis in Greece.

In order to estimate the extent of neonatal candidiasis in Greece and to identify trends in clinical management, the present study was conducted using a questionnaire directed to Greek neonatologists and pediatric infectious disease specialists. The respondents represented 15 hospitals in the country's seven largest cities, which are currently the only Greek cities with neonatal intensive care units. Based on the responses, the incidence of neonatal candidiasis was determined to be 1.87 and 1.94 cases per unit-year for the years 2001 and 2002, respectively. Although a shift toward the isolation of non-Candida albicans isolates was noted, C. albicans was still the predominant pathogenic species. Deoxycholate amphotericin B remains the most frequently used antifungal agent in neonatal units nationwide.

Neonatal candidiasis: analysis of epidemiology, drug susceptibility, and molecular typing of causative isolates.

A prospective observational study of invasive candidiasis was conducted in the neonatal intensive care unit of Aristotle University in Hippokration Hospital between 1994 and 2000. During this period, 59 neonates developed invasive candidiasis (58 cases of candidemia and 1 case of peritonitis), resulting in an overall incidence of 1.28% that showed a decreasing trend over the study period. Eleven (18.6%) cases developed within the first week of life and the others within a mean (+/-SEM) of 13.4+/-1.7 days after birth. The three most frequent causative species were Candida albicans (65.5%), Candida parapsilosis (15.5%), and Candida tropicalis (7%). C. albicans was the predominant species between 1994 and 1998, whereas, non-albicans Candida spp., particularly C. parapsilosis, were the most frequent species during the period 1999-2000 (P<0.001). While the overall mortality due to candidemia was 29% (17 of 59 cases), mortality associated with C. albicans and C. parapsilosis was 39.5% and 11.1%, respectively (P=0.032), and that observed in the 1999-2000 period was 0% (P=0.011). Virtually all isolates were susceptible to amphotericin B, flucytosine, fluconazole, and itraconazole, and no increases in minimal inhibitory concentrations were observed during these years. With the exception of a limited cluster of cases due to genotypically identical isolates, no clonal relation of C. albicans isolates was found. Moreover, no clonal persistence of C. albicans and no decrease in antifungal drug susceptibility occurred over the 6-year study period. Non-albicans Candida spp., mostly C. parapsilosis, have emerged as important pathogens in neonatal intensive care units, with infected patients having better outcomes as compared to patients infected with C. albicans.

Effects of macrophage colony-stimulating factor on antifungal activity of neonatal monocytes against Candida albicans.

The effects of recombinant macrophage-colony stimulating factor (M-CSF) on antifungal activities of monocytes (MNC) from healthy neonates and adults against Candida albicans were compared. Pretreatment of adult and neonatal MNC with 15 ng/ml of M-CSF for 4 days significantly increased superoxide anion (O(-2)) production in response to phorbol myristate acetate. While M-CSF-treated MNC from adults produced significantly higher O(-2) in response to Candida blastoconidia, M-CSF-treated neonatal MNC did not show a similar response. Further, M-CSF significantly enhanced phagocytosis of C. albicans by adult MNC but not by neonatal MNC. While M-CSF enhances antifungal activities of adult MNC against C. albicans, it does not appear to affect anticandidal function of neonatal MNC.

Flow cytometric measurement of HLA-DR expression on circulating monocytes in healthy and sick neonates using monocyte negative selection.

UNLABELLED: The aim of this study was to investigate the effect of prematurity, neonatal sepsis, respiratory distress syndrome (RDS) and perinatal asphyxia on monocyte HLA-DR expression of neonates using a flow cytometric method based on monocyte negative selection. The subjects were one hundred and thirty-one neonates (59 healthy, 44 septicaemic, 20 with RDS and eight with perinatal asphyxia) and 20 healthy adults. Monocyte HLA-DR expression was measured using one-colour HLA-DR labelling in a gate for monocytes obtained using the combination of CD3-CD19--PE/CD15--FITC MoAbs. In addition, the common dual staining method using MoAbs against two CD14 epitopes (TUK4, MO2) was evaluated. With the one-colour HLA-DR labelling higher purity and recovery values of monocytes were achieved than with the dual labelling METHOD: Healthy neonates had significantly lower percentages of HLA-DR(+) monocytes than adults (69 +/- 13% versus 91.5 +/- 2.5%) and comparable mean fluorescence intensity (MFI) (119 +/- 25 versus 131 +/- 26). Values did not differ significantly between healthy term and preterm neonates. Preterm neonates with RDS had a significantly lower percentage of HLA-DR(+) monocytes than the healthy preterm neonates. In neonates with asphyxia both parameters were comparable to those of the healthy ones. Septicaemic neonates presented significantly lower values of both parameters than the healthy, RDS and asphyxiated neonates. Monocyte negative selection provides a reliable estimation of HLA-DR expression on monocytes. Expression of monocyte HLA-DR is lower in healthy neonates in comparison with adults and is further decreased in neonates with sepsis and RDS, but it is not influenced by prematurity and perinatal asphyxia.

Evidence of early systemic activation and transendothelial migration of neutrophils in neonates with severe respiratory distress syndrome.

Several observations imply that the early inflammatory response involving activated neutrophils, tissue macrophages, and cytokines plays an important role in the pathogenesis of neonatal respiratory distress syndrome (RDS) and progression to bronchopulmonary dysplasia (BPD). The aim of this study was to test the hypothesis that changes in circulating neutrophil number and function and plasma levels of cytokines, consistent with neutrophil activation and migration to the tissues, occur during the early stages of neonatal RDS. For this purpose we measured peripheral blood levels of certain immunological parameters that promote neutrophil activation and transendothelial migration. Twenty preterm neonates with severe RDS and 20 healthy infants matched for gestational age were the subjects. The absolute neutrophil count (ANC), and plasma levels of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and sL-selectin using an enzyme-linked immunosorbent assay (ELISA), neutrophil CD11b expression, and respiratory burst activity (RBA) using flow cytometry, were measured within 24 h after birth. The two groups were comparable regarding perinatal characteristics. None of the neonates studied had any clinical or laboratory evidence of infection by the time of blood sampling. The immunological investigation showed that the RDS neonates had significantly lower ANC (P = 0.032), higher expression of the CD11b on neutrophils (P = 0.0065), and higher G-CSF and IL-6 plasma levels (P = 0.0047 and P < 0.0001, respectively) in comparison to healthy preterm neonates. The neutrophil RBA and plasma sL-selectin levels did not differ significantly between the two groups. We conclude that in neonates with severe RDS, there is evidence of a systemic neutrophil activation early in the course of the disease, supporting the view of a contributing role of activated neutrophils in the pathogenesis of RDS.

Use of erythropoietin and its effects on blood lactate and 2, 3-diphosphoglycerate in premature neonates.

The aim of this study was to investigate the effect of recombinant human erythropoietin (rHu-EPO) on oxygen affinity and adequate oxygen delivery to the tissues of stable premature infants. 36 very-low-birth-weight infants were randomly assigned to either receive rHu-EPO (200 units/kg every other day) or not, and both groups were supplemented with iron, folic acid and vitamin E. Arterial blood gases, oxygen saturation, complete blood counts, fetal haemoglobin, 2,3-diphosphoglycerate (2,3-DPG) and blood lactate were analysed weekly, from the 1st week till discharge. Patients in the two groups were comparable. There was a trend in increasing lactate values towards the 4th to 5th weeks of life, which did not reach statistical significance. There was no correlation between lactate values and the studied variables (pH, BE, oxygen saturation). In 35 transfusions, pre- and 24 h post-transfusion blood lactate status was studied. In 23 of them, a decrease in post-transfusion lactate was noticed, whilst an increased post-transfusion level was shown in 10 cases and no change in 2 cases. The mean pre-transfusion lactate value was significantly higher than the post-transfusion one (24.04 +/- 11.9 mg/dl before and 16.27 +/- 8.5 mg/dl after transfusion; p = 0.0025). In both groups there was a steady rise in 2,3-DPG concentration over the period of study, and the 2,3-DPG values at the end of our study were significantly increased in the rHu-EPO group (rHu-EPO 5.98 +/- 0.9, control 4.84 +/- 0.7; p = 0.04). In conclusion, the use of rHu-EPO did not affect blood lactate levels compared to the control group. Regarding oxygen affinity, it seems that rHu-EPO causes a shift of the oxy-haemoglobin dissociation curve to the right. This is a previously unreported effect of rHu-EPO and its clinical use may, thus, confer to preterm babies an added advantage.

Septicemia due to multiresistant Klebsiella pneumoniae in a neonatal unit: a case-control study.

An unusually high incidence of septicemia due to multiresistant Klebsiella pneumoniae occurred in the Aristotle University Neonatology Department. Forty neonates suffered from 42 episodes of septicemia. Mortality was 43% ranging from 32% in neonates with birth weight (BW) - 1,500 g to 55% with < 1,500 g. No differences were found between 17 neonates who died and 23 survivors. All isolates were resistant to aminoglycosides, third-generation cephalosporins, and aztreonam, but susceptible to imipenem and ciprofloxacin. The neonates with septicemia due to K. pneumoniae were matched 1:1 with neonates without septicemia (31 pairs) or with neonates with septicemia due to other organisms (8 pairs) according to BW and time of admission. Factors associated with septicemia were mechanical ventilation (p = 0.004) and ongoing parenteral nutrition (p = 0.027). In a multivariate model, nutrition exhibited no independent association after adjusting for ventilation. No differences were detected between the patients with Klebsiella septicemia and those with septicemia due to other organisms. Enhanced Infection Control measures and a temporary change of antibiotic policy reduced this serious complication. Three small outbreaks of multiresistant K. pneumoniae previously reported in neonates are reviewed.

Plantar response profile of high-risk infants at one year of life.

To clarify the plantar reflex profile at 1 year of life in different categories of neurodevelopmental abnormalities, plantar responses were examined prospectively in 204 high-risk infants, of whom 58 developed cerebral palsy, 22 had developmental retardation without motor disturbance, and 124 were normal at a follow-up examination at 3 years of age. The plantar response was extensor in 82.3% of infants subsequently found to be neurologically normal at the first month of life, becoming flexor at the age of 9 and 11 months in 68.5% and 86.3%, respectively. Twenty-one (42.9%) of 49 patients with various types of spastic cerebral palsy demonstrated a combined extensor response (ie, dorsiflexion of the great toe with fanning of the remaining toes) as early as the first month of life. Children with spastic quadriplegia and hemiplegia more frequently demonstrated a combined extensor response compared to diplegic patients. The combined extensor plantar response remains a reliable prognostic clinical tool that contributes to an earlier diagnosis of spastic cerebral palsy as early as the first month of life.

Influence of recombinant human erythropoietin on neutrophil function in premature neonates.

The in vivo influence of recombinant human erythropoietin (rhEpo) and iron on human neutrophil (PMN) antimicrobial function was assessed. A total of 21 preterm infants were randomized to receive either 200 U/kg/other day of rHuEPO+12 mg/kg/day of iron (EPO+high Fe, seven infants) or 200 U/kg/other day of rhEPO+4 mg/kg/day of iron (EPO+standard Fe, 9 infants) or 4 mg/kg/day of iron only (standard Fe, five infants). PMNs were isolated from blood of these infants 60+/-5 days after birth and from eight healthy adults. No differences between infants and adults were found in PMN random migration and chemotactic activity to N-formylmethionyl leucyl phenylalanine (FMLP), superoxide anion production in response to FMLP and phagocytosis of Staphylococcus aureus. In contrast, percentage phagocytosis was significantly lower in EPO+standard Fe as compared to both EPO+high Fe and standard Fe groups (P<0.01). This modest impairment of phagocytic activity of neonatal PMNs found in association with administration of rhEPO and standard iron may be related to consumption of iron during rhEPO-enhanced erythropoiesis.

Using postural reactions as a screening test to identify high-risk infants for cerebral palsy: a prospective study.

To clarify the predictive value of the seven more commonly used postural reactions (PR) in the 1st year of life regarding the diagnosis of cerebral palsy (CP), we prospectively examined 204 high-risk infants of whom 58 developed CP, 22 had developmental retardation (DR) and 124 were normal at follow-up at 3 years of age. Abnormalities of five or more PR from the 1st month of life were correlated with spastic CP, while five or six abnormal PR were also correlated with athetoid CP. Three or less abnormal PR correlated with a normal outcome. All seven PR tested were significantly abnormal in children with spastic CP from the 1st month compared to normal children. Athetoid children demonstrated abnormalities of the Peiper-Isbert (P-I) reaction and Vojta reaction from the 1st month and of the vertical, horizontal and Collis vertical suspension from the 3rd month. Children with DR had significantly abnormal Collis horizontal and Collis vertical suspension, Vojta reaction and traction response from the 1st month and Peiper-Isbert reaction from the 3rd month. Ataxic children demonstrated significantly abnormal traction response from the 1st month, Collis horizontal reaction from the 7th month and Peiper-Isbert reaction from the 11th month. We conclude that the examination of PR is a useful quantitative and qualitative diagnostic screening tool for high-risk infants from the 1st month of life.

Administration of recombinant human granulocyte-colony stimulating factor to septic neonates induces neutrophilia and enhances the neutrophil respiratory burst and beta2 integrin expression. Results of a randomized controlled trial.

The objective of this study was to investigate the effect of treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the neutrophil count and function of preterm neonates with documented sepsis. For this purpose 62 preterm neonates with proven sepsis and 19 healthy preterm ones were studied. Of the 62 patients, 27 septic neonates had an absolute neutrophil count (ANC) > 5000/mm3 (group A) and were scheduled not to receive rhG-CSF and 35/62 had an ANC < 5000/mm3 (n=35) and were randomly assigned either to receive rhG-CSF (group B) or not to receive it (group C). rhG-CSF (10 microg/kg) was administered for 3 consecutive days (0, 1, 2). The ANC, plasma levels of G-CSF (ELISA), neutrophil respiratory burst activity (NRBA) and neutrophil expression of CD11a, CD11b and CD11c (flow cytometry) were measured in all septic neonates on days 0 (onset of sepsis), 1, 3 and 5 and in the healthy neonates once within the first 2 days of life. We found that on day 0, G-CSF levels of all groups of septic neonates were significantly higher than those of the healthy ones. The highest levels were observed in group A. NRBA was diminished only in groups B and C and the expression of CD11a and CD11c was reduced in all groups of septic neonates. Administration of rhG-CSF resulted in a rapid and significant increase in ANC, NRBA and CD11a, CD11b and CD11c expression that persisted throughout the follow up. CONCLUSION; The administration of granulocyte colony stimulating factor to septic neonates significantly increases the absolute granulocyte count and enhances the neutrophil respiratory burst and beta2 integrin expression.

Multifocal osteoarthritis due to Candida albicans in a neonate: serum level monitoring of liposomal amphotericin B and literature review.

A 32-week neonate weighing 2,300 g at birth with fungemia due to Candida albicans subsequently developed multifocal osteoarthritis of the lower extremities due to the same organism during therapy with amphotericin B (0.5 mg/kg/day) and flucytosine (100 mg/kg/day) to which the isolates were susceptible. Liposomal amphotericin B (3.5 mg/kg/day) was substituted for conventional amphotericin B and complete clinical and radiologic recovery as well as sterilization of affected joints were achieved with a 38-day-course (144.5 mg/kg total). Adequate drug concentrations in serum and synovial fluid were attained with liposomal amphotericin B. Nine neonates (< or = 28 days of age) with bone and/or joint infections due to Candida spp. had previously been reported in the English-language literature. To our knowledge, the present case is the first reported cure with liposomal amphotericin B. Previous cases are reviewed and the potential role of liposomal amphotericin B in this serious neonatal infection is discussed.

Impact of prematurity, stress and sepsis on the neutrophil respiratory burst activity of neonates.

The aim of this study was to evaluate the impact of prematurity, sepsis and stress on the neutrophil respiratory burst activity (NRBA) of neonates. For this purpose 122 healthy neonates (89 term and 33 preterm), 33 preterm stressed neonates, 59 septic neonates (12 term and 47 preterm) and 26 healthy adults were studied. The NRBA was assessed after in vitro stimulation by PMA using a whole blood flow cytometric microassay with dihydrorhodamine 123 (DHR 123). It was found that the percentage of responding neutrophils in term neonates was comparable to that found in adults (medians 83.5 and 89.8%, respectively), whereas it was significantly lower in the healthy preterm neonates (median 70.6%, p < 0.05). The NRBA was further depressed in the stressed (median = 63%) and septic neonates, both term and preterm (medians 60.5 and 54.3%, respectively). No correlation with the levels of G-CSF, TNF-alpha and IL-1 beta, which were found to be higher in the stressed and septic neonates, was observed. These findings indicate that prematurity, sepsis and stress significantly depress the respiratory burst activity of neonatal neutrophils.

Prospective follow-up of primitive reflex profiles in high-risk infants: clues to an early diagnosis of cerebral palsy.

To clarify reflex profiles in the first year of life in connection with categories of neurologic abnormality, eight primitive reflexes (i.e., the palmar grasp reflex, the plantar grasp reflex, the Galant response, the asymmetric tonic neck reflex, the suprapubic extensor reflex, the crossed extensor reflex, the Rossolimo reflex, and the heel reflex) were prospectively examined in 204 high-risk infants, of whom 58 developed cerebral palsy, 22 had developmental retardation, and 124 were normal at follow-up examination at 2 years of age. The change in the retention time of reflex activity for each of these reflexes was characteristic for each category or type of neurologic abnormality: retention of palmar grasp reflex, suprapubic extensor reflex, crossed extensor reflex, Rossolimo reflex, and heel reflex in spastic cerebral palsy, as well as retention of plantar grasp reflex, Galant reflex, and asymmetric tonic neck reflex in athetoid cerebral palsy and somewhat weaker retention of these reflexes in developmental retardation (statistical significance P < .001 compared with normally developed patients). These characteristic changes imply that a presumptive diagnosis can be made in neurologically high-risk infants by examination of the primitive reflexes, which are of specific significance among the other neurologic criteria within the first year of life.

Follow-up of very low birth weight infants after erythropoietin treatment to prevent anemia of prematurity.

OBJECTIVE: Treatment with recombinant human erythropoietin (rHuEPO) stimulates erythropoiesis and reduces the need for transfusions in hospitalized preterm infants. The aim of our study was to follow very low birth weight infants after the initial 6 weeks of rHuEPO treatment. DESIGN AND METHODS: We randomly assigned 97 very low birth weight infants with a gestational age of 31 weeks or less and birth weight of 1500 gm or less to receive rHuEPO, 300 units/kg per week (erythropoietin (EPO) 300, n = 33), rHuEPO, 750 units/kg per week (EPO 750; n = 28), or no treatment (control, n = 36). The rHuEPO was administered from the first week of life for 6 weeks. After EPO therapy was discontinued, 75 neonates were followed weekly until discharge and at 3, 6, and 12 months of age. RESULTS: Mean numbers (+/- SD) of packed erythrocyte transfusions per patient from the time rHuEPO therapy was discontinued until discharge were 0.38 +/- 0.64 (EPO 300), 0.23 +/- 0.52 (EPO 750), 0.9 +/- 1.1 (control) (p < 0.05 in both EPO groups vs control). Mean reticulocyte counts at the sixth week were 6% +/- 2.2% (EPO 300), 6.9% +/- 2.2% (EPO 750), and 3.1% +/- 2.6% (control) in the three groups (p < 0.01 in both EPO groups vs control), and at the eighth week were 4.7% +/- 2.8% (EPO 300), 5.4% +/- 2.7% (EPO 750), and 2.6% +/- 2.2% (control) (p < 0.01 in both EPO groups vs control). Serum ferritin levels were significantly higher at the sixth week, and the percentage of hemoglobin F was significantly lower at 6, 8, and 10 weeks in the control group versus EPO groups. At 3, 6, and 12 months of age, there were no differences in reticulocytes, ferritin, HbF, and growth among groups. CONCLUSION: Preterm infants who received rHuEPO had a normal pattern of erythropoiesis after the drug was discontinued. These data provide strong evidence that the anemia of prematurity is the result of a transient developmental abnormality in EPO production.

Concentrations of main serum opsonins in early infancy.

The evolution of the main serum opsonins in neonates and infants of varying gestational age was investigated to provide reference values for these opsonins in early infancy. Serum concentrations of immunoglobulins, IgG subclasses, C3, C4 and fibronectin were serially measured from birth until the age of 6 months in term and preterm infants. Measurements were performed by rate nephelometry. Five hundred and sixty six neonates (gestational age 26-41 weeks) were examined at birth, 233 at 1 month, 218 at 3 months, and 147 at 6 months, respectively. The same measurements were performed in 54 pairs of neonatal/maternal samples and in 230 apparently healthy adults. Gestational age had a significant impact on serum IgG, IgG subclasses, C3 and C4 up till the third month, and on fibronectin until the first month. No such impact was observed for IgA and IgM. Sixteen per cent of the neonates had IgM concentrations higher than 0.2 g/l at birth, suggesting that the critical concentration of serum IgM at birth for suspected intrauterine infection should be reconsidered. Concentrations of all opsonins at birth were significantly lower than adult reference values. They only approached or even reached adult values by the third or the sixth month. Data from analysis of the neonatal and the corresponding maternal sera indicate that there is a preferential active transplacental transport of IgG subclasses in the order of IgG1, IgG3, IgG2 and IgG4. These results show that concentrations of immunoglobulins, C3, C4 and fibronectin undergo changes during the first months of life, depending not only on the infants' postnatal age but also on gestational age.

Serum concentrations of 10 acute-phase proteins in healthy term and preterm infants from birth to age 6 months.

Aiming to define the evolution pattern of 10 acute-phase proteins in early infancy, we measured nephelometrically the serum concentrations of albumin, prealbumin, retinol-binding protein, transferrin, ceruloplasmin, hemopexin, haptoglobin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, and alpha 1-antitrypsin in 395 term and preterm infants (gestational ages 26-41 weeks). Measurements were performed within 24 h after birth and then at the end of 1 (n = 171), 3 (n = 155), and 6 (n = 90) months afterwards. Data obtained from 250 healthy adults were used as adult reference values. All proteins increased progressively with postnatal age, except for alpha 1-antitrypsin, which remained stable from birth to the 6th month. Concentrations of almost all measured proteins were significantly lower in preterm than in term infants in the first 3 months. Compared with adult values, alpha 2-macroglobulin and alpha 1-antitrypsin were higher in infants throughout the 6 months. The other proteins were significantly lower at birth than adult values but after 6 months, only albumin, prealbumin, retinol-binding protein, and alpha 1-acid glycoprotein still remained lower in infants. Thus both gestational and postnatal age should be considered when interpreting concentrations of these proteins in early infancy.

In which neonates does early recombinant human erythropoietin treatment prevent anemia of prematurity? Results of a randomized, controlled study.

To assess whether erythropoietin (EPO) treatment is safe and reduces the need for transfusion, we randomized 44 preterm infants to an EPO group and a comparable control (CON) group. EPO 150 U/kg was given s.c. twice weekly for 6 wk from the 1st wk of life. Hematologic parameters, transfusion requirements, and growth were followed during therapy and for 6 mo thereafter. To better assess in which neonates EPO treatment was effective, we classified retrospectively the EPO and CON groups into uncomplicated neonates (EPO A: n = 9, birth weight = 1247 +/- 126 g, gestational age = 29.8 +/- 1.5 wk; CON A: n = 7, birth weight = 1217 +/- 145 g, gestational age = 29.9 +/- 1.5 wk) and neonates requiring artificial ventilation (EPO B: n = 16, birth weight = 1169 +/- 249 g, gestational age = 28.1 +/- 2 wk; CON B: n = 12, birth weight = 1173 +/- 215 g, gestational age = 28.3 +/- 2 wk). There were significant differences in reticulocytes between both uncomplicated and ventilated neonates in the EPO group compared with respective control groups. However, the need for transfusion was significantly less in the uncomplicated EPO group (EPO A: 0.44 +/- 0.73 versus CON A: 1.28 +/- 0.75, p < 0.05) but not in the neonates on ventilation (EPO B: 8.25 +/- 5 versus CON B: 7.75 +/- 3.7). In conclusion, early EPO administration reduces the need for transfusion in uncomplicated premature neonates.(ABSTRACT TRUNCATED AT 250 WORDS)