Scandinavian Journal of Infectious Diseases, 2014; 46: 433–439
ORIGINAL ARTICLE
Diagnostic accuracy and prognostic value of the CD64 index in very low birth weight neonates as a marker of early-onset sepsis
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MARIO MOTTA1, ALICE ZINI1, ANTONIO REGAZZOLI2, ELENA GARZOLI3, GAETANO CHIRICO1, LUIGI CAIMI2 & MARINELLA CALARCO2 From the 1Neonatology and Neonatal Intensive Care Unit, Children’s Hospital of Brescia, 2Laboratorio Chimica-Clinica, Spedali Civili Brescia, Brescia, and 3Neonatology and Neonatal Intensive Care Unit, Azienda Ospedaliera “Papa Giovanni XXIII”, Bergamo, Italy
Abstract Objective: To assess the diagnostic and prognostic utility of CD64 expression as a marker of early-onset sepsis (EOS) in very low birth weight (VLBW) neonates. Methods: Neutrophil CD64 expression (CD64 index) was assessed in 129 VLBW neonates within 72 h after birth. The accuracy of the CD64 index in predicting EOS was determined by receiver operating characteristic curve analysis. The relationship between the expression of the CD64 index and neonatal outcomes was evaluated by multivariate analysis. Results: The highest performance of the CD64 index was achieved at 24 h after birth; accuracy, sensitivity, and negative predictive values were 0.85, 0.89, and 0.99, respectively, with a cut-off value of 2.4. The increased expression of CD64 index was significantly associated with subsequent infections (relative risk 1.54; 95% confidence interval 1.02–2.33). Conclusions: The CD64 index could be used as a reliable marker of EOS in VLBW neonates and it is an independent risk factor for late-onset infections.
Keywords: Newborn, sepsis, IgG receptors
Introduction The most common cause of death in the neonatal period is preterm birth, followed by severe infections [1]. In neonates, early-onset sepsis (EOS) is a systemic infection acquired by vertical transmission from the mother, typically occurring during the first 72 h after birth [2]. Maternal prolonged rupture of the membranes, chorioamnionitis or intrauterine infection, and preterm delivery are the most documented risk factors for neonatal EOS [3–5]. There is growing evidence showing the link between intrauterine infection and preterm delivery, as well as adverse neonatal outcomes including perinatal death, respiratory distress, chronic lung disease, brain injury, and long-term disability [6–8]. The inflammatory cascade triggered by intrauterine infection appears to be one of the main factors
involved in the pathogenesis of prematurity-related complications; this clinical setting is termed foetal inflammatory response syndrome [9]. The up-regulation of the neutrophil CD64 molecule, the high-affinity receptor in the family of FCγ receptors, has been reported in neonates with bacterial infections, thus indicating that the measurement of neutrophil CD64 expression at birth correlates with the presence of EOS [10–14]. This prospective study aimed to: (1) evaluate the diagnostic accuracy of early postnatal expression of neutrophil CD64 assessment (within 72 h from birth) for the identification of EOS in very low birth weight (VLBW) neonates, and (2) to evaluate the relationship between the expression of neutrophil CD64 and the occurrence of neonatal mortality and morbidities in VLBW neonates.
Correspondence: M. Motta, Neonatology and Neonatal Intensive Care, Children’s Hospital of Brescia, P. le Spedali Civili, 25123 Brescia, Italy. Tel: ⫹ 39 0303995219. Fax: ⫹ 39 0303700817. E-mail: [email protected] (Received 1 August 2013 ; accepted 18 January 2014 ) ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2014.896028
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Materials and methods
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Patients VLBW neonates with a postnatal age of ⱕ 72 h, admitted to the neonatal intensive care unit (NICU) of the Children’s Hospital of Brescia, Italy, were eligible for enrolment into the study. Neonates who had congenital or chromosomal abnormalities were excluded. The recruitment of neonates was conducted prospectively over a 24-month period, from July 2009 to June 2011. Neonates recruited into the study were observed over a period of 72 h from birth and were divided into 2 groups according to their infection screening: (1) The sepsis group comprised neonates with suspected or proven EOS. Suspected sepsis was diagnosed in the presence of a clinical suspicion of neonatal infection (including temperature instability, hyperglycaemia (defined as values ⬎ 150 mg/dl), unexplained tachycardia, respiratory distress, and neurological compromise) combined with a positive C-reactive protein (defined as values ⬎ 10 mg/l) with the support of an immature/total neutrophil ratio of ⬎ 0.2 at blood count [15], and/or positive surface (ears, nose, throat, and umbilicus), urine, gastric aspirate, stool, and endotracheal aspirate specimens for potential pathogen culture. Proven sepsis was defined as a positive blood culture associated with clinical and laboratory features suggestive of neonatal sepsis as reported above. (2) The noninfected group comprised neonates with negative microbial screening cultures, including blood culture, without clinical and laboratory signs suggestive of infection as described above. Blood cultures were obtained from all neonates included in the study at the time of NICU admission. In detail, a single 1.0-ml sample of peripheral blood was drawn using standard sterile techniques in a single paediatric blood culture bottle (BACTEC Peds Plus/F Culture Vials; Becton Dickinson, Franklin Lakes, NJ, USA). The data of neonates included in the study were collected using an electronic database (Microsoft Excel, Redmond, WA, USA); the following were recorded: demographic characteristics, clinical and laboratory values, and the occurrence of neonatal complications. Neonatal complications included the following: mortality, late-onset sepsis (LOS), patent ductus arteriosus (PDA), intraventricular haemorrhage (IVH), periventricular leukomalacia (PVL), retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD) defined as oxygen dependence at 36 weeks of postmenstrual age. Cases of LOS were considered those neonates with
suspected or proven sepsis at a postnatal age of more than 72 h with clinical and laboratory criteria as specified for EOS. IVH and PVL were diagnosed by cranial sonography and graded using the classification of Papile et al. and de Vries et al., respectively [16,17]. Cases of PDA were considered those who required pharmacological or surgical treatment. ROP was diagnosed according to the International Classification of Retinopathy of Prematurity [18]. NEC was diagnosed according to the classification of Bell et al. [19].
Blood sampling and CD64 index assessment Blood samples (0.5 ml) were obtained from all neonates at birth at the time of NICU admission together with samples for routine laboratory tests. A second blood sample was obtained between 25 and 72 h after birth from those neonates who required additional laboratory tests. The expression of CD64 and CD163 on neutrophils, monocytes, and lymphocytes was measured by quantitative flow cytometry with a Cytomics FC500 (Beckman Coulter, Miami, FL, USA) using the Leuko64 assay (Leuko64 kit; Trillium Diagnostics, Scarborough, ME, USA). This assay is composed of a mixture of 3 murine monoclonal antibodies with specificities to CD64 (clones 22 and 32.2, both fluorescein isothiocyanate conjugated) and to CD163 (clone Mac2–158, phycoerythrin conjugated), and a triple-fluorescence bead suspension (green, orange, and red fluorescence) used for instrument calibration and standardization of leukocyte CD64 and CD163 expression in human blood. Fifteen microlitres of whole blood were incubated for 10 min at room temperature with the mixture of murine monoclonal antibodies, followed by ammonium chloride-based red cell lysis. Five microlitres of fluorescence beads were then added and flow cytometer analysis was performed on a minimum of 50 ⫻ 103 leukocytes. The mean fluorescence intensity was measured as a linearized value of log scale on lymphocytes (negative control), monocytes (positive control), neutrophils and beads using light scatter gating. The results of the Leuko64 assay are reported as the CD64 index of polymorphonuclear leukocytes. Index calculation was performed by Leuko64 QuantiCalc software (Trillium Diagnostics, Scarborough, ME, USA) and was derived from the ratio of linearized mean fluorescence intensity of the neutrophil population to the fluorescence signal from the beads. The internal negative and positive controls of the assay were provided by the automated measurement of the lymphocyte and monocyte CD64 indexes, respectively.
CD64 index as a marker of EOS in neonates
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Statistical analysis
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The study was approved by the Ethics Committee of the Children’s Hospital, Spedali Civili of Brescia. Written informed consent was obtained from the parents of all neonates included in the study.
58 detections obtained on day 1 and at 25–72 h after birth, respectively) were included in the study. Among these 129 VLBW neonates, 48 were included in the sepsis group (4 proven and 44 suspected EOS) and 81 were included in the noninfected group. The characteristics of neonates are summarized in Table I. Neonates included in the sepsis group had a significantly lower gestational age and birth weight than non-infected neonates. The occurrence of maternal chorioamnionitis was significantly higher in the neonates in the sepsis group compared to the group of non-infected neonates. Neonates in the sepsis group had significantly higher neutrophil CD64 indexes, C-reactive protein levels, and immature/total neutrophil ratios, and significantly lower platelet counts and haematocrit levels than noninfected neonates (Table I). Twenty-eight and 27 neonates from the sepsis group and from the noninfected group, respectively, had paired samples taken at 0–24 and 25–72 h after birth. The values of the CD64 index obtained at 25–72 h after birth were significantly higher than the values obtained at 0–24 h after birth, in both the septic and non-infected neonate groups (Table I). Neonates in the sepsis group more frequently required catecholamine infusion, mechanical ventilation, and surfactant administration compared to non-infected neonates (Table I). Logistic regression analysis showed a significant association between EOS and the CD64 index, with a relative risk of 2.21 (95% CI 1.56–3.13), independent of gestational age, birth weight, and maternal chorioamnionitis (Table II). Results of the ROC curve analysis according to the day of CD64 index detection are reported in Table III. The highest performance of the CD64 index was achieved for 25–72 h after birth, reaching accuracy, sensitivity, and negative predictive values of 0.85, 0.89 and 0.99, respectively, with a CD64 index cut-off value of 2.4. According to logistic regression analysis, gestational age was the only independent variable associated with a higher risk of neonatal mortality (Table IV). In addition, as expected, lower gestational age correlated with the occurrence of LOS, PDA, IVH, ROP, and BPD (Table IV). Among independent variables, the CD64 index was significantly associated with an increased risk of LOS (Table IV).
Results
Discussion
During the study period, 217 VLBW neonates satisfied the inclusion criteria; however, 88 of them were lost because it was not possible to determine the CD64 index in the first 72 h after birth or parental consent was denied. Therefore, 129 VLBW neonates with a total of 187 CD64 index detections (129 and
In this study, the evaluation of the CD64 index was focused on VLBW neonates since this population is at higher risk of EOS and related complications. According to their infection screening at birth, 2 groups of VLBW neonates were compared and, as expected, neonates included in the sepsis group were
The characteristics of the 2 groups of neonates were analysed using descriptive statistics. Continuous variables were presented as the median and interquartile range (IQR), and were compared by Mann– Whitney U-test. Categorical variables were presented as absolute number and percentage, and were compared using a non-parametric test (Chi-square test or Fisher’s exact test). Receiver operating characteristic (ROC) curve analysis was used to estimate the accuracy of the CD64 index in predicting EOS among VLBW neonates. The overall test performance was expressed as the area under the ROC curve with 95% confidence interval (CI). The associated Youden index, corresponding to the threshold value of the CD64 index for which both sensitivity and specificity are maximized values, was determined and the related positive and negative predictive values were calculated [20]. More specifically, a prevalence value of 3.8%, corresponding to the local area-based average value of proven EOS, was used to calculate the positive and negative predictive values. Logistic regression analysis was done to evaluate the possible association between the CD64 index and the occurrence of EOS independently from those variables that were significantly different between septic and non-infected neonates in the previously performed univariate analysis. Finally, a logistic regression analysis was used to evaluate the possible association between the continuous independent variables of gestational age, birth weight, and CD64 expression, and the occurrence of neonatal complications as categorical dependent variables. Computations were performed using 2 commercial statistical packages: SPSS for Windows (SPSS Inc., Chicago, IL, USA) and MedCalc for Windows (MedCalc Software, Mariakerke, Belgium). Differences were considered statistically significant at a p-value of ⬍ 0.05. Ethical approval
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M. Motta et al. Table I. Characteristics of very low birth weight neonates according to their infection screening at birth.
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Characteristics of neonates Gestational age, weeks, median; IQRa Gestational complications Pregnancy-induced hypertension Diabetes Clinical chorioamnionitisb,c P-PROM IUGR Birth weight, g, median; IQRa Male:female (n) CD64 index, median; IQR 0–72 h after birtha 0–24 h after birthd 25–72 h after birthd CRP, mg/l, median; IQRa WBC, ⫻ 106/ml, median; IQR ANC, ⫻ 106/ml, median; IQR Immature/total neutrophil ratio, median; IQRa PLT, ⫻ 106/ml, median; IQRc Ht, %, median; IQRe Need of catecholamine infusion, n (%)e Need of mechanical ventilation, n (%)a Need of surfactant administration, n (%)a
Sepsis group (n ⫽ 48)
Non-infected group (n ⫽ 81)
27.1; 25.3–28.2
30.0; 28.6–32.2
14 1 7 9 8 810; 583–1010 30:18
16 2 2 10 16 1135; 925–1297 50:31
2.67; 1.64–4.31 1.61; 1.35–2.64 3.4; 2.60–4.61 17.7; 12.5–25.4 6.5; 3.9–12.8 3.1; 1.3–6.6 0.32; 0.24–0.43 156; 118–208 42; 39–45 43 (89%) 48 (100%) 41 (85%)
1.45; 1.10–1.98 1.17; 0.87–1.48 2.03; 1.36–2.76 0.9; 0.7–3.5 8.0; 6.1–10.8 3.3; 2.0–6.5 0.07; 0.04–0.12 172; 144–230 45; 42–50 48 (59%) 48 (59%) 41 (50%)
IQR, interquartile range; P-PROM, preterm premature rupture of membranes; IUGR, intrauterine growth restriction; CRP, C-reactive protein; WBC, white blood cells; ANC, absolute neutrophil count; PLT, platelets; Ht, haematocrit. ap ⬍ 0.001 between the sepsis group and non-infected group. bClinical chorioamnionitis was diagnosed by the presence of a temperature elevation to 37.8°C or higher and 2 or more of the following criteria: uterine tenderness, malodorous vaginal discharge, foetal tachycardia (heart rate ⬎ 160 beats/min), maternal tachycardia (heart rate ⬎ 100 beats/min), and maternal leukocytosis (leukocyte count ⬎ 15.0 cells ⫻ 106/ml) [21]. cp ⬍ 0.05 between the sepsis group and non-infected group. dp ⬍ 0.01 within 0–24 vs 25–72 h after birth and between the sepsis group and non-infected group. ep ⬍ 0.01 between the sepsis group and non-infected group.
significantly less mature and had lower birth weights than non-infected neonates. In addition, neonates in the sepsis group, if compared to non-infected neonates, were more likely to be critically ill with a significantly higher need of cardio-respiratory support. In previous studies, discordant results on CD64 neutrophil expression in patients of different ages have been obtained. Payne et al. showed a decreased expression of CD64 on polymorphonuclear leukocytes in extremely premature neonates compared to Table II. Association between the characteristics of very low birth weight neonates and the occurrence of early-onset sepsis by logistic regression analysis.
Characteristics of neonates
p-Value
Relative risk
Gestational age Birth weight Maternal chorioamnionitis CD64 index
0.062 0.003 0.121 ⬍ 0.001
0.834 0.997 3.853 2.210
95% Confidence interval 0.680–1.003 0.995–0.987 0.702–21.270 1.560–3.130
term neonates [22]. In the study of Maeda et al., neonates expressed significantly higher CD64 levels on granulocytes compared to adults [23]. In contrast, Fjaertoft et al. observed increased levels of neutrophil CD64 expression in preterm neonates compared to term neonates and adults [24], while no statistically significant difference in neutrophil CD64 expression was found between preterm and term neonates in the study of Dilli et al [25]. In our study, by univariate analysis, the neutrophil expression of CD64 was significantly higher in neonates in the sepsis group compared to non-infected neonates (Table I). In addition, regression analysis showed a significant association between the occurrence of EOS and the expression of CD64 independent of the gestational age at birth (Table II). These findings could suggest that this test would be appropriate to rule out EOS in VLBW neonates independent of their condition of prematurity. In the current study, an increasing level of neutrophil CD64 expression was observed from birth to
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Table III. Accuracy and Youden index with related coordinates of CD64 index ROC curve, according to the time of detection during the first 72 h after birth in very low birth weight neonates. Time of CD64 detection 0–72 h after birth 0–24 h after birth 25–72 h after birth
AUC (95% CI)
Youden index value
Sensitivity (95% CI)
Specificity (95% CI)
PPV (95% CI)
NPV (95% CI)
0.76 (0.70–0.82) 0.73 (0.64–0.82) 0.85 (0.75–0.92)
2.26
0.65 (0.54–0.76) 0.58 (0.42–0.74) 0.89 (0.75–0.97)
0.85 (0.77–0.91) 0.79 (0.67–0.88) 0.76 (0.61–0.87)
0.15 (0.05–0.32) 0.10 (0.01–0.30) 0.13 (0.02–0.35)
0.98 (0.95–1.00) 0.98 (0.92–0.99) 0.99 (0.93–1.00)
1.7 2.4
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ROC, receiver operating characteristic; AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.
72 h after birth. The highest performance of the CD64 index was achieved 24 h after birth, exhibiting a good sensitivity (0.89) and a high negative predictive value (0.99) for a CD64 index cut-off value of 2.4 (Table III). Similarly, in the study of Ng et al., neutrophil expression of CD64 demonstrated its highest levels 24 h after the onset of EOS (0.97 sensitivity and 0.98 negative predictive value) [10]. In the same way, in the study by Groselj-Grenc et al., the best performance of the CD64 index was again observed 24 h after the time of suspected infection (0.96, 0.96, and 0.95 for accuracy, sensitivity, and negative predictive value, respectively) [26]. All together the results of these studies suggest that a single detection of CD64 neutrophil expression could be a reliable diagnostic marker of EOS when performed 24 h after birth. This would be particularly important in VLBW preterm neonates where EOS is a common condition and the possibility of ruling out an infection at birth could significantly affect the therapeutic decision in view of antibiotic treatment. In contrast, in our study a low specificity and low positive predictive value were obtained for the CD64 neutrophil expression in predicting EOS (Table III),
thus limiting its possible use to minimize the unnecessary antibiotic treatment of false-positive cases. In addition, it is important to note that since neutrophil CD64 expression has been evaluated differently in the different reported studies, a comparison of neutrophil CD64 cut-off values is not possible at this time. Therefore, standardization of the method should be addressed in further studies on the use of neutrophil CD64 expression as a marker of neonatal sepsis. With regard to the prognostic perspective, as expected, the current study confirmed that gestational age was the most important factor associated with neonatal mortality and morbidities. In addition, increased neutrophil CD64 expression at birth was found to be an independent risk factor of LOS in this study, with a relative risk of 1.5 (Table IV). This finding is in contrast to that of a recent study by Wynn et al. in which a significant association between earlyand late-onset sepsis in VLBW neonates was not demonstrated [27]. However, in the study of Wynn et al., only neonatal cases of blood culture-confirmed sepsis were included and, considering the low sensitivity of blood culture for the diagnosis of neonatal EOS, this
Table IV. Association between the characteristics of neonates at birth and the occurrence of neonatal complications by logistic regression analysis. Characteristics of neonates at birth Gestational age Neonatal complications
p-Value
Mortality 0.003 LOS 0.001 PDA ⬍ 0.001 IVH grade 1 or 2 ⬍ 0.001 IVH grade 3 or 4 0.043 PVL 0.207 ROP 0.001 NEC 0.878 BPD ⬍ 0.001
Birth weight
CD64 index
RR
95% CI
p-Value
RR
95% CI
p-Value
RR
95% CI
0.691 0.650 0.553 0.504 0.670 0.781 0.340 1.019 0.392
0.540–0.880 0.490–0.850 0.410–0.730 0.370–0.690 0.450–0.989 0.530–1.150 0.181–0.642 0.750–1.403 0.238–0.624
0.186 0.013 0.287 0.935 0.472 0.760 0.336 0.613 0.002
0.997 0.996 0.999 1.000 0.998 0.999 0.996 0.997 0.995
0.996–0.999 0.994–0.998 0.997–1.001 0.998–1.002 0.995–1.001 0.995–1.002 0.991–1.001 0.994–1.000 0.993–0.998
0.310 0.042 0.992 0.532 0.318 0.872 0.645 0.336 0.458
0.852 1.540 1.00 0.90 1.20 1.04 1.12 1.22 1.20
0.629–1.161 1.020–2.330 0.723–1.402 0.649–1.242 0.830–1.753 0.662–1.649 0.681–1.840 0.824–1.820 0.730–1.989
RR, relative risk; CI, confidence interval; LOS, late-onset sepsis; PDA, patent ductus arteriosus; IVH, intraventricular haemorrhage; PVL, periventricular leukomalacia; ROP, retinopathy of prematurity; NEC, necrotizing enterocolitis; BPD, bronchopulmonary dysplasia.
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test would not be the best predictor of LOS. In contrast, a more sensitive test, such as the increased expression of neutrophil CD64, could be a better predictor of subsequent infections. The association between EOS and LOS can be explained by the observation that in animal models, during early life, high levels of pro-inflammatory signals would be likely to interfere with the normal process of immune development, inducing a subsequent deficient or abnormal response to infections [28]. Moreover, thymus and spleen depletion have been reported in human neonates who have died because of EOS associated with chorioamnionitis, suggesting that immune function impairment in this clinical setting is secondary to the early systemic inflammatory response [29,30]. The significant association between the CD64 index and LOS should be considered with interest, as this test, when performed 24 h after birth, could identify a subset of VLBW neonates at an increased risk of subsequent infection who may receive specific treatment to improve their prognosis. A possible limitation of this study, with regard to the accuracy of the CD64 index for the identification of EOS, was the inclusion of many cases of suspected sepsis without a positive blood culture, which remains the gold standard for the diagnosis of neonatal sepsis. This low rate of positive blood culture is probably related to the characteristics of our study population. In this study, blood culture was obtained early after birth in VLBW neonates. In this clinical setting, blood contamination from maternal antibiotic treatment and the limited blood volume available for culture (1 ml) may have been determining factors in reducing the sensitivity of blood culture. Of practical importance is that since a substantial proportion of neutrophils undergoes apoptosis at 24 h after blood collection and down-regulates the cell surface antigens, our study blood specimens were processed only if assessment of neutrophil CD64 expression would have been possible within 8 h from blood collection. Given that during the study period the assessment of neutrophil CD64 expression by flow cytometry was possible only on working days, this was the main cause of the high number of dropout cases. In conclusion, in this study the early postnatal assessment of the CD64 index (from 24 to 72 h after birth) showed a good performance in predicting neonatal EOS. In addition, the CD64 index was proven to be an independent risk factor for LOS in VLBW neonates. Declaration of interest: The authors disclaim any competing interest. The authors have not received any honorarium, grant, or other form of payment for producing the manuscript.
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ORIGINAL ARTICLE
Diagnostic accuracy and prognostic value of the CD64 index in very low birth weight neonates as a marker of early-onset sepsis
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MARIO MOTTA1, ALICE ZINI1, ANTONIO REGAZZOLI2, ELENA GARZOLI3, GAETANO CHIRICO1, LUIGI CAIMI2 & MARINELLA CALARCO2 From the 1Neonatology and Neonatal Intensive Care Unit, Children’s Hospital of Brescia, 2Laboratorio Chimica-Clinica, Spedali Civili Brescia, Brescia, and 3Neonatology and Neonatal Intensive Care Unit, Azienda Ospedaliera “Papa Giovanni XXIII”, Bergamo, Italy
Abstract Objective: To assess the diagnostic and prognostic utility of CD64 expression as a marker of early-onset sepsis (EOS) in very low birth weight (VLBW) neonates. Methods: Neutrophil CD64 expression (CD64 index) was assessed in 129 VLBW neonates within 72 h after birth. The accuracy of the CD64 index in predicting EOS was determined by receiver operating characteristic curve analysis. The relationship between the expression of the CD64 index and neonatal outcomes was evaluated by multivariate analysis. Results: The highest performance of the CD64 index was achieved at 24 h after birth; accuracy, sensitivity, and negative predictive values were 0.85, 0.89, and 0.99, respectively, with a cut-off value of 2.4. The increased expression of CD64 index was significantly associated with subsequent infections (relative risk 1.54; 95% confidence interval 1.02–2.33). Conclusions: The CD64 index could be used as a reliable marker of EOS in VLBW neonates and it is an independent risk factor for late-onset infections.
Keywords: Newborn, sepsis, IgG receptors
Introduction The most common cause of death in the neonatal period is preterm birth, followed by severe infections [1]. In neonates, early-onset sepsis (EOS) is a systemic infection acquired by vertical transmission from the mother, typically occurring during the first 72 h after birth [2]. Maternal prolonged rupture of the membranes, chorioamnionitis or intrauterine infection, and preterm delivery are the most documented risk factors for neonatal EOS [3–5]. There is growing evidence showing the link between intrauterine infection and preterm delivery, as well as adverse neonatal outcomes including perinatal death, respiratory distress, chronic lung disease, brain injury, and long-term disability [6–8]. The inflammatory cascade triggered by intrauterine infection appears to be one of the main factors
involved in the pathogenesis of prematurity-related complications; this clinical setting is termed foetal inflammatory response syndrome [9]. The up-regulation of the neutrophil CD64 molecule, the high-affinity receptor in the family of FCγ receptors, has been reported in neonates with bacterial infections, thus indicating that the measurement of neutrophil CD64 expression at birth correlates with the presence of EOS [10–14]. This prospective study aimed to: (1) evaluate the diagnostic accuracy of early postnatal expression of neutrophil CD64 assessment (within 72 h from birth) for the identification of EOS in very low birth weight (VLBW) neonates, and (2) to evaluate the relationship between the expression of neutrophil CD64 and the occurrence of neonatal mortality and morbidities in VLBW neonates.
Correspondence: M. Motta, Neonatology and Neonatal Intensive Care, Children’s Hospital of Brescia, P. le Spedali Civili, 25123 Brescia, Italy. Tel: ⫹ 39 0303995219. Fax: ⫹ 39 0303700817. E-mail: [email protected] (Received 1 August 2013 ; accepted 18 January 2014 ) ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2014.896028
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Materials and methods
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Patients VLBW neonates with a postnatal age of ⱕ 72 h, admitted to the neonatal intensive care unit (NICU) of the Children’s Hospital of Brescia, Italy, were eligible for enrolment into the study. Neonates who had congenital or chromosomal abnormalities were excluded. The recruitment of neonates was conducted prospectively over a 24-month period, from July 2009 to June 2011. Neonates recruited into the study were observed over a period of 72 h from birth and were divided into 2 groups according to their infection screening: (1) The sepsis group comprised neonates with suspected or proven EOS. Suspected sepsis was diagnosed in the presence of a clinical suspicion of neonatal infection (including temperature instability, hyperglycaemia (defined as values ⬎ 150 mg/dl), unexplained tachycardia, respiratory distress, and neurological compromise) combined with a positive C-reactive protein (defined as values ⬎ 10 mg/l) with the support of an immature/total neutrophil ratio of ⬎ 0.2 at blood count [15], and/or positive surface (ears, nose, throat, and umbilicus), urine, gastric aspirate, stool, and endotracheal aspirate specimens for potential pathogen culture. Proven sepsis was defined as a positive blood culture associated with clinical and laboratory features suggestive of neonatal sepsis as reported above. (2) The noninfected group comprised neonates with negative microbial screening cultures, including blood culture, without clinical and laboratory signs suggestive of infection as described above. Blood cultures were obtained from all neonates included in the study at the time of NICU admission. In detail, a single 1.0-ml sample of peripheral blood was drawn using standard sterile techniques in a single paediatric blood culture bottle (BACTEC Peds Plus/F Culture Vials; Becton Dickinson, Franklin Lakes, NJ, USA). The data of neonates included in the study were collected using an electronic database (Microsoft Excel, Redmond, WA, USA); the following were recorded: demographic characteristics, clinical and laboratory values, and the occurrence of neonatal complications. Neonatal complications included the following: mortality, late-onset sepsis (LOS), patent ductus arteriosus (PDA), intraventricular haemorrhage (IVH), periventricular leukomalacia (PVL), retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD) defined as oxygen dependence at 36 weeks of postmenstrual age. Cases of LOS were considered those neonates with
suspected or proven sepsis at a postnatal age of more than 72 h with clinical and laboratory criteria as specified for EOS. IVH and PVL were diagnosed by cranial sonography and graded using the classification of Papile et al. and de Vries et al., respectively [16,17]. Cases of PDA were considered those who required pharmacological or surgical treatment. ROP was diagnosed according to the International Classification of Retinopathy of Prematurity [18]. NEC was diagnosed according to the classification of Bell et al. [19].
Blood sampling and CD64 index assessment Blood samples (0.5 ml) were obtained from all neonates at birth at the time of NICU admission together with samples for routine laboratory tests. A second blood sample was obtained between 25 and 72 h after birth from those neonates who required additional laboratory tests. The expression of CD64 and CD163 on neutrophils, monocytes, and lymphocytes was measured by quantitative flow cytometry with a Cytomics FC500 (Beckman Coulter, Miami, FL, USA) using the Leuko64 assay (Leuko64 kit; Trillium Diagnostics, Scarborough, ME, USA). This assay is composed of a mixture of 3 murine monoclonal antibodies with specificities to CD64 (clones 22 and 32.2, both fluorescein isothiocyanate conjugated) and to CD163 (clone Mac2–158, phycoerythrin conjugated), and a triple-fluorescence bead suspension (green, orange, and red fluorescence) used for instrument calibration and standardization of leukocyte CD64 and CD163 expression in human blood. Fifteen microlitres of whole blood were incubated for 10 min at room temperature with the mixture of murine monoclonal antibodies, followed by ammonium chloride-based red cell lysis. Five microlitres of fluorescence beads were then added and flow cytometer analysis was performed on a minimum of 50 ⫻ 103 leukocytes. The mean fluorescence intensity was measured as a linearized value of log scale on lymphocytes (negative control), monocytes (positive control), neutrophils and beads using light scatter gating. The results of the Leuko64 assay are reported as the CD64 index of polymorphonuclear leukocytes. Index calculation was performed by Leuko64 QuantiCalc software (Trillium Diagnostics, Scarborough, ME, USA) and was derived from the ratio of linearized mean fluorescence intensity of the neutrophil population to the fluorescence signal from the beads. The internal negative and positive controls of the assay were provided by the automated measurement of the lymphocyte and monocyte CD64 indexes, respectively.
CD64 index as a marker of EOS in neonates
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Statistical analysis
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The study was approved by the Ethics Committee of the Children’s Hospital, Spedali Civili of Brescia. Written informed consent was obtained from the parents of all neonates included in the study.
58 detections obtained on day 1 and at 25–72 h after birth, respectively) were included in the study. Among these 129 VLBW neonates, 48 were included in the sepsis group (4 proven and 44 suspected EOS) and 81 were included in the noninfected group. The characteristics of neonates are summarized in Table I. Neonates included in the sepsis group had a significantly lower gestational age and birth weight than non-infected neonates. The occurrence of maternal chorioamnionitis was significantly higher in the neonates in the sepsis group compared to the group of non-infected neonates. Neonates in the sepsis group had significantly higher neutrophil CD64 indexes, C-reactive protein levels, and immature/total neutrophil ratios, and significantly lower platelet counts and haematocrit levels than noninfected neonates (Table I). Twenty-eight and 27 neonates from the sepsis group and from the noninfected group, respectively, had paired samples taken at 0–24 and 25–72 h after birth. The values of the CD64 index obtained at 25–72 h after birth were significantly higher than the values obtained at 0–24 h after birth, in both the septic and non-infected neonate groups (Table I). Neonates in the sepsis group more frequently required catecholamine infusion, mechanical ventilation, and surfactant administration compared to non-infected neonates (Table I). Logistic regression analysis showed a significant association between EOS and the CD64 index, with a relative risk of 2.21 (95% CI 1.56–3.13), independent of gestational age, birth weight, and maternal chorioamnionitis (Table II). Results of the ROC curve analysis according to the day of CD64 index detection are reported in Table III. The highest performance of the CD64 index was achieved for 25–72 h after birth, reaching accuracy, sensitivity, and negative predictive values of 0.85, 0.89 and 0.99, respectively, with a CD64 index cut-off value of 2.4. According to logistic regression analysis, gestational age was the only independent variable associated with a higher risk of neonatal mortality (Table IV). In addition, as expected, lower gestational age correlated with the occurrence of LOS, PDA, IVH, ROP, and BPD (Table IV). Among independent variables, the CD64 index was significantly associated with an increased risk of LOS (Table IV).
Results
Discussion
During the study period, 217 VLBW neonates satisfied the inclusion criteria; however, 88 of them were lost because it was not possible to determine the CD64 index in the first 72 h after birth or parental consent was denied. Therefore, 129 VLBW neonates with a total of 187 CD64 index detections (129 and
In this study, the evaluation of the CD64 index was focused on VLBW neonates since this population is at higher risk of EOS and related complications. According to their infection screening at birth, 2 groups of VLBW neonates were compared and, as expected, neonates included in the sepsis group were
The characteristics of the 2 groups of neonates were analysed using descriptive statistics. Continuous variables were presented as the median and interquartile range (IQR), and were compared by Mann– Whitney U-test. Categorical variables were presented as absolute number and percentage, and were compared using a non-parametric test (Chi-square test or Fisher’s exact test). Receiver operating characteristic (ROC) curve analysis was used to estimate the accuracy of the CD64 index in predicting EOS among VLBW neonates. The overall test performance was expressed as the area under the ROC curve with 95% confidence interval (CI). The associated Youden index, corresponding to the threshold value of the CD64 index for which both sensitivity and specificity are maximized values, was determined and the related positive and negative predictive values were calculated [20]. More specifically, a prevalence value of 3.8%, corresponding to the local area-based average value of proven EOS, was used to calculate the positive and negative predictive values. Logistic regression analysis was done to evaluate the possible association between the CD64 index and the occurrence of EOS independently from those variables that were significantly different between septic and non-infected neonates in the previously performed univariate analysis. Finally, a logistic regression analysis was used to evaluate the possible association between the continuous independent variables of gestational age, birth weight, and CD64 expression, and the occurrence of neonatal complications as categorical dependent variables. Computations were performed using 2 commercial statistical packages: SPSS for Windows (SPSS Inc., Chicago, IL, USA) and MedCalc for Windows (MedCalc Software, Mariakerke, Belgium). Differences were considered statistically significant at a p-value of ⬍ 0.05. Ethical approval
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M. Motta et al. Table I. Characteristics of very low birth weight neonates according to their infection screening at birth.
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Characteristics of neonates Gestational age, weeks, median; IQRa Gestational complications Pregnancy-induced hypertension Diabetes Clinical chorioamnionitisb,c P-PROM IUGR Birth weight, g, median; IQRa Male:female (n) CD64 index, median; IQR 0–72 h after birtha 0–24 h after birthd 25–72 h after birthd CRP, mg/l, median; IQRa WBC, ⫻ 106/ml, median; IQR ANC, ⫻ 106/ml, median; IQR Immature/total neutrophil ratio, median; IQRa PLT, ⫻ 106/ml, median; IQRc Ht, %, median; IQRe Need of catecholamine infusion, n (%)e Need of mechanical ventilation, n (%)a Need of surfactant administration, n (%)a
Sepsis group (n ⫽ 48)
Non-infected group (n ⫽ 81)
27.1; 25.3–28.2
30.0; 28.6–32.2
14 1 7 9 8 810; 583–1010 30:18
16 2 2 10 16 1135; 925–1297 50:31
2.67; 1.64–4.31 1.61; 1.35–2.64 3.4; 2.60–4.61 17.7; 12.5–25.4 6.5; 3.9–12.8 3.1; 1.3–6.6 0.32; 0.24–0.43 156; 118–208 42; 39–45 43 (89%) 48 (100%) 41 (85%)
1.45; 1.10–1.98 1.17; 0.87–1.48 2.03; 1.36–2.76 0.9; 0.7–3.5 8.0; 6.1–10.8 3.3; 2.0–6.5 0.07; 0.04–0.12 172; 144–230 45; 42–50 48 (59%) 48 (59%) 41 (50%)
IQR, interquartile range; P-PROM, preterm premature rupture of membranes; IUGR, intrauterine growth restriction; CRP, C-reactive protein; WBC, white blood cells; ANC, absolute neutrophil count; PLT, platelets; Ht, haematocrit. ap ⬍ 0.001 between the sepsis group and non-infected group. bClinical chorioamnionitis was diagnosed by the presence of a temperature elevation to 37.8°C or higher and 2 or more of the following criteria: uterine tenderness, malodorous vaginal discharge, foetal tachycardia (heart rate ⬎ 160 beats/min), maternal tachycardia (heart rate ⬎ 100 beats/min), and maternal leukocytosis (leukocyte count ⬎ 15.0 cells ⫻ 106/ml) [21]. cp ⬍ 0.05 between the sepsis group and non-infected group. dp ⬍ 0.01 within 0–24 vs 25–72 h after birth and between the sepsis group and non-infected group. ep ⬍ 0.01 between the sepsis group and non-infected group.
significantly less mature and had lower birth weights than non-infected neonates. In addition, neonates in the sepsis group, if compared to non-infected neonates, were more likely to be critically ill with a significantly higher need of cardio-respiratory support. In previous studies, discordant results on CD64 neutrophil expression in patients of different ages have been obtained. Payne et al. showed a decreased expression of CD64 on polymorphonuclear leukocytes in extremely premature neonates compared to Table II. Association between the characteristics of very low birth weight neonates and the occurrence of early-onset sepsis by logistic regression analysis.
Characteristics of neonates
p-Value
Relative risk
Gestational age Birth weight Maternal chorioamnionitis CD64 index
0.062 0.003 0.121 ⬍ 0.001
0.834 0.997 3.853 2.210
95% Confidence interval 0.680–1.003 0.995–0.987 0.702–21.270 1.560–3.130
term neonates [22]. In the study of Maeda et al., neonates expressed significantly higher CD64 levels on granulocytes compared to adults [23]. In contrast, Fjaertoft et al. observed increased levels of neutrophil CD64 expression in preterm neonates compared to term neonates and adults [24], while no statistically significant difference in neutrophil CD64 expression was found between preterm and term neonates in the study of Dilli et al [25]. In our study, by univariate analysis, the neutrophil expression of CD64 was significantly higher in neonates in the sepsis group compared to non-infected neonates (Table I). In addition, regression analysis showed a significant association between the occurrence of EOS and the expression of CD64 independent of the gestational age at birth (Table II). These findings could suggest that this test would be appropriate to rule out EOS in VLBW neonates independent of their condition of prematurity. In the current study, an increasing level of neutrophil CD64 expression was observed from birth to
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Table III. Accuracy and Youden index with related coordinates of CD64 index ROC curve, according to the time of detection during the first 72 h after birth in very low birth weight neonates. Time of CD64 detection 0–72 h after birth 0–24 h after birth 25–72 h after birth
AUC (95% CI)
Youden index value
Sensitivity (95% CI)
Specificity (95% CI)
PPV (95% CI)
NPV (95% CI)
0.76 (0.70–0.82) 0.73 (0.64–0.82) 0.85 (0.75–0.92)
2.26
0.65 (0.54–0.76) 0.58 (0.42–0.74) 0.89 (0.75–0.97)
0.85 (0.77–0.91) 0.79 (0.67–0.88) 0.76 (0.61–0.87)
0.15 (0.05–0.32) 0.10 (0.01–0.30) 0.13 (0.02–0.35)
0.98 (0.95–1.00) 0.98 (0.92–0.99) 0.99 (0.93–1.00)
1.7 2.4
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ROC, receiver operating characteristic; AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.
72 h after birth. The highest performance of the CD64 index was achieved 24 h after birth, exhibiting a good sensitivity (0.89) and a high negative predictive value (0.99) for a CD64 index cut-off value of 2.4 (Table III). Similarly, in the study of Ng et al., neutrophil expression of CD64 demonstrated its highest levels 24 h after the onset of EOS (0.97 sensitivity and 0.98 negative predictive value) [10]. In the same way, in the study by Groselj-Grenc et al., the best performance of the CD64 index was again observed 24 h after the time of suspected infection (0.96, 0.96, and 0.95 for accuracy, sensitivity, and negative predictive value, respectively) [26]. All together the results of these studies suggest that a single detection of CD64 neutrophil expression could be a reliable diagnostic marker of EOS when performed 24 h after birth. This would be particularly important in VLBW preterm neonates where EOS is a common condition and the possibility of ruling out an infection at birth could significantly affect the therapeutic decision in view of antibiotic treatment. In contrast, in our study a low specificity and low positive predictive value were obtained for the CD64 neutrophil expression in predicting EOS (Table III),
thus limiting its possible use to minimize the unnecessary antibiotic treatment of false-positive cases. In addition, it is important to note that since neutrophil CD64 expression has been evaluated differently in the different reported studies, a comparison of neutrophil CD64 cut-off values is not possible at this time. Therefore, standardization of the method should be addressed in further studies on the use of neutrophil CD64 expression as a marker of neonatal sepsis. With regard to the prognostic perspective, as expected, the current study confirmed that gestational age was the most important factor associated with neonatal mortality and morbidities. In addition, increased neutrophil CD64 expression at birth was found to be an independent risk factor of LOS in this study, with a relative risk of 1.5 (Table IV). This finding is in contrast to that of a recent study by Wynn et al. in which a significant association between earlyand late-onset sepsis in VLBW neonates was not demonstrated [27]. However, in the study of Wynn et al., only neonatal cases of blood culture-confirmed sepsis were included and, considering the low sensitivity of blood culture for the diagnosis of neonatal EOS, this
Table IV. Association between the characteristics of neonates at birth and the occurrence of neonatal complications by logistic regression analysis. Characteristics of neonates at birth Gestational age Neonatal complications
p-Value
Mortality 0.003 LOS 0.001 PDA ⬍ 0.001 IVH grade 1 or 2 ⬍ 0.001 IVH grade 3 or 4 0.043 PVL 0.207 ROP 0.001 NEC 0.878 BPD ⬍ 0.001
Birth weight
CD64 index
RR
95% CI
p-Value
RR
95% CI
p-Value
RR
95% CI
0.691 0.650 0.553 0.504 0.670 0.781 0.340 1.019 0.392
0.540–0.880 0.490–0.850 0.410–0.730 0.370–0.690 0.450–0.989 0.530–1.150 0.181–0.642 0.750–1.403 0.238–0.624
0.186 0.013 0.287 0.935 0.472 0.760 0.336 0.613 0.002
0.997 0.996 0.999 1.000 0.998 0.999 0.996 0.997 0.995
0.996–0.999 0.994–0.998 0.997–1.001 0.998–1.002 0.995–1.001 0.995–1.002 0.991–1.001 0.994–1.000 0.993–0.998
0.310 0.042 0.992 0.532 0.318 0.872 0.645 0.336 0.458
0.852 1.540 1.00 0.90 1.20 1.04 1.12 1.22 1.20
0.629–1.161 1.020–2.330 0.723–1.402 0.649–1.242 0.830–1.753 0.662–1.649 0.681–1.840 0.824–1.820 0.730–1.989
RR, relative risk; CI, confidence interval; LOS, late-onset sepsis; PDA, patent ductus arteriosus; IVH, intraventricular haemorrhage; PVL, periventricular leukomalacia; ROP, retinopathy of prematurity; NEC, necrotizing enterocolitis; BPD, bronchopulmonary dysplasia.
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test would not be the best predictor of LOS. In contrast, a more sensitive test, such as the increased expression of neutrophil CD64, could be a better predictor of subsequent infections. The association between EOS and LOS can be explained by the observation that in animal models, during early life, high levels of pro-inflammatory signals would be likely to interfere with the normal process of immune development, inducing a subsequent deficient or abnormal response to infections [28]. Moreover, thymus and spleen depletion have been reported in human neonates who have died because of EOS associated with chorioamnionitis, suggesting that immune function impairment in this clinical setting is secondary to the early systemic inflammatory response [29,30]. The significant association between the CD64 index and LOS should be considered with interest, as this test, when performed 24 h after birth, could identify a subset of VLBW neonates at an increased risk of subsequent infection who may receive specific treatment to improve their prognosis. A possible limitation of this study, with regard to the accuracy of the CD64 index for the identification of EOS, was the inclusion of many cases of suspected sepsis without a positive blood culture, which remains the gold standard for the diagnosis of neonatal sepsis. This low rate of positive blood culture is probably related to the characteristics of our study population. In this study, blood culture was obtained early after birth in VLBW neonates. In this clinical setting, blood contamination from maternal antibiotic treatment and the limited blood volume available for culture (1 ml) may have been determining factors in reducing the sensitivity of blood culture. Of practical importance is that since a substantial proportion of neutrophils undergoes apoptosis at 24 h after blood collection and down-regulates the cell surface antigens, our study blood specimens were processed only if assessment of neutrophil CD64 expression would have been possible within 8 h from blood collection. Given that during the study period the assessment of neutrophil CD64 expression by flow cytometry was possible only on working days, this was the main cause of the high number of dropout cases. In conclusion, in this study the early postnatal assessment of the CD64 index (from 24 to 72 h after birth) showed a good performance in predicting neonatal EOS. In addition, the CD64 index was proven to be an independent risk factor for LOS in VLBW neonates. Declaration of interest: The authors disclaim any competing interest. The authors have not received any honorarium, grant, or other form of payment for producing the manuscript.
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