PEDIATRICS Vol. 107 No. 1 January 2001, pp. 30-35

A Randomized, Double-Masked, Placebo-Controlled Trial of Recombinant Granulocyte Colony-Stimulating Factor Administration to Preterm Infants With the Clinical Diagnosis of Early-Onset Sepsis

Ernani Miura, MD, PhD^*, Renato S. Procianoy, MD^*, Cristina Bittar, MD^*, Clarissa S. Miura, MD^*, Maurício S. Miura^*, Cíntia Mello, MD^*, and Robert D. Christensen, MD

From the ^* Department of Pediatrics, Division of Neonatology, Hospital de Clínicas de Porto Alegre, Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil; and the  Department of Pediatrics, Division of Neonatology, University of Florida College of Medicine, Gainesville, Florida.

	ABSTRACT

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Objective.  We performed a randomized, double-masked, parallel-groups, placebo-controlled trial of recombinant granulocyte colony-stimulating factor (rG-CSF) administration to 44 preterm neonates who had blood cultures obtained and antibiotics begun because of the clinical diagnosis of early-onset sepsis. Two primary outcome variables were tested 1) mortality and 2) development of nosocomial infections over the 2-week period after dosing.

Design and Methods.  The treatment group (n = 22) received 10 µg/kg/day of intravenous rG-CSF once daily for 3 days and the placebo group (n = 22) received the same volume of a visually indistinguishable vehicle. Mortality and culture-proven nosocomial infections were recorded. Immediately before the first, second, and third doses, and again 10 days after the first dose, serum concentrations were determined for tumor necrosis factor-, interleukin 6, granulocyte-macrophage colony stimulating factor, and G-CSF, and blood leukocyte counts, absolute neutrophil counts, immature/total neutrophil ratios, platelet counts, and hemoglobin concentrations were measured.

Results.  The treatment and placebo groups were of similar gestational age (29 ± 3 vs 31 ± 3 weeks) and birth weight (1376 ± 491 vs 1404 ± 508 g), and had similar Apgar scores and 24-hour Score for Neonatal Acute Physiology scores. The mortality rate was not different between treatment and placebo groups. However, the occurrence of a subsequent nosocomial infection was lower in the rG-CSF recipients (relative risk: .19; 95% confidence interval: .05-.78). rG-CSF treatment did not alter the serum concentrations of the cytokines measured (except for G-CSF). Serum G-CSF levels and blood neutrophil counts were higher in the treatment than in the placebo group 24 hours and 48 hours after dosing.

Conclusions.  Administration of 3 daily doses of rG-CSF (10 µg/kg/day) to premature neonates with the clinical diagnosis of early-onset sepsis did not improve mortality but was associated with acquiring fewer nosocomial infections over the subsequent 2 weeks.  Key words:  granulocyte colony-stimulating factor, neutropenia, early-onset sepsis, absolute neutrophil count, neutrophil storage pool, neutrophil proliferative pool..

Neonatal host defense is characterized by immaturity of select aspects of humoral and cellular immunity.^1,2 Qualitative and quantitative deficiencies of neutrophils are reproducible findings and likely contribute to the mortality and morbidity from neonatal sepsis.^1-5

Granulocyte colony-stimulating factor (G-CSF) is a physiologic regulator of neutrophil production and function. Its actions include clonal maturation of neutrophil progenitors and enhancement of mature neutrophil functions, including chemotaxis, phagocytosis, superoxide production, and bactericidal activity.^6,7 Production of G-CSF by monocytes from newborn infants is relatively poor, compared with monocytes from adults.^8-10 Also, plasma concentrations of G-CSF are lower in neutropenic neonates than in comparably neutropenic adults.^8-11 These findings have fostered the speculation that G-CSF production is somewhat limited in neonates and that recombinant (r)G-CSF administration might improve the outcome of infected neonates or might reduce their incidence of nosocomial infection.^8-11 Indeed, these have been shown in neonatal animals with experimental infections.^12-14 Moreover, a phase I/II study of rG-CSF administration to human neonates with suspected sepsis showed a dose-dependent increase in circulating and storage neutrophils,¹⁵ and 3 clinical trials have reported variable effects of rG-CSF on survival from sepsis.^16-18 However, these 3 all involved rather small groups of subjects, and 2 used concurrent or historical controls, rather than using a randomized, double-masked, parallel groups, placebo-controlled design.

We hypothesized that the administration of 3 daily doses of G-CSF (10 µg/kg/day) to premature newborn infants with the clinical diagnosis of early-onset sepsis would: 1) reduce mortality and 2) reduce the subsequent acquisition of a nosocomial infection for a period of 2 weeks after the dosing. The rationale for hypothesizing a mortality rate reduction was the enhancement of antibacterial capacity resulting from quantitative and qualitative changes in neutrophils induced by rG-CSF. The rational for limiting the proposed protection from nosocomial infection to 2 weeks was the unlikelihood that the salutary effects of rG-CSF on neutrophil quantity or quality would persist >10 to 14 days after dosing.

	METHODS

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Patient Selection Criteria

From July 1996 to July 1997, we evaluated for possible study entry all neonates weighing 500 to 2000 g, with a gestational age <37 weeks, who were <5 days old, at the Neonatal Unit of Hospital de Clínicas de Porto Alegre, with a clinical diagnosis of early-onset bacterial sepsis. The clinical diagnosis of sepsis was defined according to the report of Gillan et al¹⁵ as "having a blood culture obtained and antibiotic treatment begun for suspected bacterial sepsis." To improve uniformity of the study subjects, 2 additional entry criteria were required. Specifically, we required the presence of 3 or more of the following clinical criteria and 1 or more of the following laboratory criteria. In order for a criterion to be counted, it was necessary that the item be unexplained, except by sepsis. For instance, if hypotension had a reasonable causation other than sepsis, then the criterion was not counted. The clinical criteria were: 1) an abnormal core body temperature not explained by environmental influences, 2) respiratory distress (including apnea [defined as 2 or more spells, lasting at least 15 seconds, associated with bradycardia or hypoxemia, detected during a period of 60 minutes], and tachypnea), 3) neurologic dysfunction, defined as either a seizure or hypotonia, 4) abdominal distention or emesis, 5) hyperbilirubinemia, 6) hypotension, and 7) diffuse hemorrhage. The laboratory criteria were: 1) neutropenia, defined as an absolute neutrophil count (ANC) <1500/µL, 2) an immature to total blood neutrophil (I/T) ratio >.2,¹⁹ 3) serum interleukin 6 (IL-6) >32 pg/mL,²⁰ and 4) serum tumor necrosis factor- (TNF-) >12 pg/mL.²¹ Patients were ineligible if their mother received 2 or more doses of antibiotics before delivery or if the neonate had severe congenital malformations or severe asphyxia at birth without response to resuscitation measures.

Nosocomial infections were defined by: 1) a positive blood culture, obtained because of clinical deterioration consistent with a nosocomial infection after the fifth day of life or 2) radiographically (pneumatosis intestinalis) or pathologically confirmed necrotizing enterocolitis (NEC). The protocol was approved by our institutional ethics committee and for each case a written informed consent was obtained.

Treatment Protocol

All patients were treated with conventional therapeutic interventions including antibiotics, supplemental oxygen, mechanical ventilation, intravenous fluids, and vasoactive drugs as deemed necessary by the attending neonatologist. Neonates in the treatment group received intravenous rG-CSF (Amgen, Thousand Oaks, CA) at a dose of 10 µg/kg/day in 10 mL of 5% dextrose and .2% albumin (2 mg/mL) infused by a pump over 30 minutes once daily for 3 consecutive days. The placebo consisted of an identical volume of the vehicle without rG-CSF. The solutions were visually indistinguishable.

All eligible neonates were randomized to receive either rG-CSF or placebo using a predetermined schedule generated from random numbers. Participants, investigators, and health care providers were blinded to the study assignment. The dose of study medication was withheld if the ANC exceeded 60 000/µL, if any toxicity was deemed related to the administration of study medication, or if the parental consent was withdrawn.

Laboratory Evaluation

The leukocyte count (LC), ANC, I/T ratio, hemoglobin concentration, and platelet count were measured before administration of the first dose of rG-CSF or placebo and were repeated 24 hours, 72 hours, and 10 days later (the 24- and 72-hour values were obtained immediately before the second and third doses of study medication). Complete blood counts were performed electronically, and differential LCs were performed manually on May-Grunwald-Giemsa-stained blood films.

Patients underwent tibial bone marrow aspiration on the seventh day after the first dose of study medication, and differential cell counts were performed on May-Grunwald-Giemsa-stained smears. The proportion of nucleated marrow cells identified as myeloblasts, promyelocytes, or myelocytes were termed the neutrophil proliferative pool (NPP) percent, and the proportion of nucleated marrow cells identified as metamyelocytes, band neutrophils, or segmented neutrophils were termed the neutrophil storage pool (NSP) percent.

Quantification of Cytokines

Peripheral blood samples were obtained to determine the concentrations of G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and TNF-, immediately before the first administration of rG-CSF or placebo and again 24 hours, 72 hours, and again 10 days later. Samples were stored at 70°C and analyzed in batches. These cytokines were measured by standardized sandwich enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN). The linear limits of detectable concentrations for the cytokines were as follows: for G-CSF, 11 to 2500 pg/mL; for GM-CSF, 7.8 to 500 pg/mL (values were extrapolated to as low as 3 pg/mL); for IL-6, 3 to 300 pg/mL; and for TNF-, 16 to 1000 pg/mL (values were extrapolated to as low as 11 pg/mL). Serum sample dilutions were performed to enable G-CSF measurements of up to 6000 pg/mL. Any G-CSF concentrations exceeding 6000 pg/mL were reported as 6000 pg/mL.

Score for Neonatal Acute Physiology (SNAP) and Other Outcome Measures

The SNAP²² was assessed at study entry and subsequently. Early mortality (the 5-day period after randomization) and nosocomial infections during the 14 days after the final dose of the study medication were recorded as the primary outcome measures. Secondary outcome measures were length of time on a mechanical ventilator and length of time in supplemental oxygen.

Sample-Size Calculation, Interim Analysis, and Statistical Considerations

The study was designed to test the hypothesis that rG-CSF administration, on an intend-to-treat basis, would reduce the mortality rate by 50%. The prestudy sample-size calculation was based on a standard risk for mortality of preterm infants with early-onset sepsis in our institution of 64% (the high-end of the 28-67 range published by Klein and Marcy²³). A sample size of 80 (40 + 40) was calculated, based on our hypothesized 50% risk reduction (to 32% mortality) with the condition of  = .05 and a power of .80. An interim analysis was scheduled prospectively to be conducted by an oversight committee after enrollment of 40 patients. The committee was specifically charged to examine the first 40 patients for overwhelming evidence of nonefficacy, such that the study should not continue.

Demographic data were compared with a 2-tailed t test and the nonparametric Kruskal-Wallis test. Cytokines levels (G-CSF, GM-CSF, TNF-, and IL-6), complete blood counts (LC, I/T, ANC, hemoglobin, and platelets), and bone marrow counts (NSP, NPP, and NSP/NPP) were compared by analysis of variance using the nonparametric test of Kruskal-Wallis. Values of P < .05 were considered significant.

	RESULTS

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The planned interim analysis occurred when 44 patients (rather than 40 as designed) had completed the study (22 rG-CSF and 22 placebo). The mortality rate of the 2 groups was similar (Fig 1). Furthermore, the observed mortality rate was only approximately one half of that used in the sample-size calculation. The oversight committee recommended that further patient accrual be halted, because enrollment of the additional 36 patients, as set forth in the initial design, would be extremely unlikely to show the hypothesized improvement in mortality among rG-CSF recipients. Therefore, the study was closed to enrollment, and the data on the 44 patients entered were analyzed and are herein reported.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Survival rates of preterm neonates with clinical sepsis treated with rG-CSF (straight line) or placebo (dotted line).

Thirty patients were enrolled on the first day of life, 10 on the second day, and 4 on the third or fourth day of life. The 2 groups (rG-CSF vs placebo) had similar birth weights (1404 ± 508, rG-CSF vs 1376 ± 491 g, placebo; mean ± standard deviation), gestational ages (31 ± 3 vs 29 ± 3 weeks), 5-minute Apgar scores (8; range: 1-10 vs 7; 3-10), pretreatment SNAP scores (9; range: 0-23 vs 8; 3-28), and number with a pretreatment ANC <1500 (5 vs 3). All 22 patients randomized to rG-CSF completed the 3 days of study drug administration. Twenty of the 22 patients randomized to placebo treatment completed the 3 days of administration, but 2 died on the second study day (Fig 1).

All 3 early deaths in the placebo group (2 on the second study day and 1 on the third) were judged to be infection-related, but blood cultures were negative. In each of the 3, premature rupture of membranes and clinical chorioamnionitis prompted 1 dose of antenatal penicillin or ampicillin plus gentamicin. All 3 neonates had refractory hypotension, respiratory distress, temperature instability, hypoxemia, refractory metabolic acidosis, and seizures. Case 1 had a large left shift (I/T = .42) and thrombocytopenia (58 000/uL), case 2 had neutropenia (1300/µL) All 3 early deaths had intracranial hemorrhage (case 1, grade IV; cases 2 and 3, grade III). Case 2 had severe pulmonary and renal hemorrhages.

The following bacteria were isolated from blood cultures at study entry: group B streptococcus (1 case, rG-CSF group), Staphylococcus aureus (2 cases, placebo group), Staphylococcus epidermis (1 case, rG-CSF group), Escherichia coli (1 case, placebo group), and unidentified Gram-negative bacteria (1 case, placebo group). The following were the laboratory signs of clinical sepsis: rG-CSF group, elevated I/T ratio (n = 9), low ANC (n = 3), elevated IL-6 (n = 7), and elevated TNF- (n = 21); and placebo group, elevated I/T (n = 9), low ANC (n = 5), elevated IL-6 (n = 10), and elevated TNF- (n = 21).

Before the first study dose, the serum G-CSF levels were minimally to markedly higher than in previously reported noninfected neonates (Table 1). Twenty-four hours after the first study dose (immediately before the second dose) G-CSF serum concentrations were significantly higher among rG-CSF recipients than among placebo recipients. Similarly, immediately before the third dose, G-CSF concentrations were significantly higher in rG-CSF recipients. Ten days after the start of treatment, G-CSF concentrations were similar in the 2 groups (Table 1). Plasma levels of GM-CSF, TNF-, and IL-6 were not different after the administration of rG-CSF or between the treatment and placebo groups (Table 1).

Before the first study dose, LCs and ANCs were similar in the 2 groups (Table 2). However, at 24 and 48 hours, both were significantly higher in the rG-CSF recipients than in the placebo recipients. Each of the 3 cases of neutropenia in the rG-CSF group remitted by 24 hours. However, 3 of the 5 case of neutropenia in the placebo group were still neutropenic at 48 hours. Ten days after treatment, no differences in ANC were seen between the rG-CSF and placebo recipients. No differences were observed between groups with respect to changes in I/T ratio or in absolute eosinophil, lymphocyte, or platelet count. However, higher absolute monocyte counts were observed 48 hours after rG-CSF than after placebo. No differences were observed in length of time on mechanical ventilation or in length of time on supplemental oxygen.

Seven days after the last dose of study drug, bone marrow aspirates were performed. The reason for the marrow study was that 1 of the 2 outcome variables (nosocomial infection) was based on the rational that rG-CSF had an effect on marrow neutrophil quantity that persisted after cessation of treatment. Thus, the marrow aspirate on day seven provided an opportunity to examine any relationship between protection from infection and marrow neutrophil quantity. The study was performed on 36 of the 42 study subjects; 5 died before the scheduled marrow aspiration and 1 was believed to be a poor candidate for the aspiration because of his critical condition in the neonatal intensive care unit at the time. Indeed, the NSP percent and the NSP/NPP ratios were significantly higher in the rG-CSF recipients than in the placebo recipients (Table 3).

Eleven cases of nosocomial infection were detected during the 2 weeks after study dosing; 9/19 in the placebo group (47.3%) and 2/22 in the rG-CSF group (9.1%; P < .02; relative risk: .19; 95% confidence interval: .05-.78). Nine of the 11 had positive blood cultures, and 2 were confirmed cases of NEC. Both cases of NEC occurred in the placebo group. The following bacteria were isolated from patients with nosocomial infections: S aureus (4 cases, 2 in each group), S epidermis (4 cases, all placebo recipients), and Staphylococcus hemolyticus (1 case, placebo group). All cases blood cultures were reported as positive within 24 hours of plating.

Three late deaths occurred in the placebo group; all were associated with nosocomial infections. Two died with NEC, on the 22nd and 24th days of life (1 had concomitant S epidermis sepsis and 1 had pulmonary microabscesses diagnosed on postmortem examination). The third late death among the placebo recipients had intestinal obstruction and sepsis and died postoperatively on the 24th day of life. Five late deaths occurred among the rG-CSF recipients. None were associated with infections; 2 had grade 3 intraventricular hemorrhages (days 8 and 13), 2 died with pulmonary hemorrhage (day 14), and 1 died with renal failure (day 25).

	DISCUSSION

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Early-onset infection and nosocomial infection remain major contributors to mortality, morbidity, length of hospital stay, and overall cost of neonatal intensive care.²³ Immaturity of certain antibacterial defenses, particularly in the neutrophil system, contribute to this problem.^1,2 With the availability of rG-CSF and with the discovery of its roles in neutrophil production and function and with the reports that G-CSF production is relatively limited in preterm neonates, it was speculated that rG-CSF administration might be a useful adjunct in the treatment or prevention of neonatal sepsis.^8,11

It was clear from the first rG-CSF trial in neonates by Gillan et al¹⁵ that rG-CSF administration has similar biologic effects in neonates as in adults. Namely, that it induces release of neutrophils from the NSP into the circulation and subsequently causes a dose-dependent increase in neutrophil production and in circulating and storage neutrophil concentrations. Gillan et al¹⁵ studied 42 neonates who had presumed sepsis and who were treated with either placebo (n = 9) or rG-CSF in doses ranging from 1.0 to 20.0 µg/kg/day for 3 consecutive days. None of the 42 subjects died, reflecting a population composed predominantly of noncritically ill neonates.

Kocherlakota and La Gamma¹⁶ reported a study of 25 neonates: 14 rG-CSF recipients and 11 concomitant controls. Schibler et al¹⁸ randomized 20 septic, neutropenic neonates: 10 rG-CSF and 10 placebo. Barak et al¹⁷ treated 14 neonates with rG-CSF and compared their outcome with 24 historical non-G-CSF-treated controls. Consistent with the study by Schibler et al,¹⁸ but opposite with the reports by Kocherlakota and La Gamma¹⁶ and by Barak et al,¹⁷ we observed no effect of rG-CSF on overall mortality. However, none of the previous neonatal rG-CSF trials undertook a prospective determination of the effect of rG-CSF on the subsequent development of nosocomial infections. A potential salutary effect was suggested by the finding of Gillen et al¹⁵ that rG-CSF treatment resulted in a marked increase in the size of the NSP. We made the same finding in the present study, and we also observed fewer nosocomial infections among the rG-CSF recipients. We speculate that the lower incidence of infections might be the result of a larger NSP, which persisted for an undetermined period of time after cessation of the rG-CSF dosing. Alternatively, perhaps an improvement in neutrophil function, persisting for a time after cessation of the rG-CSF dosing or a combination of quantitative and qualitative neutrophil effects was mechanistically responsible.

Cairo et al²⁷reported a randomized, placebo-controlled trial among very low birth weight neonates, comparing the incidence of nosocomial infections after the prophylactic use of rGM-CSF. They studied 264 neonates <72 hours of life: 134 received 8 µg/kg/day of intravenous rGM-CSF daily for 7 days, then every other day for 21 days and 130 received placebo. In the rGM-CSF recipients, a significant increase in ANC occurred on days 7, 14, and 21. However, there was no difference in incidence of nosocomial infections between groups. Carr et al²⁸ used rGM-CSF prophylatically in a randomized, controlled trial among 75 premature neonates during the first 72 hours of life and found that neutropenia was abolished in the treated group, compared with a 40% incidence in the controls. There was no difference in mortality between the groups, but the treated group had a tendency toward fewer nosocomial infections.

Reports by Gessler et al,²⁴ Kennon et al,²⁵ and Calhoun et al²⁶ indicate that neonates with overwhelming sepsis generally have high endogenous G-CSF concentrations, but our patients tended to have only moderately elevated pretreatment G-CSF concentrations. Perhaps this signifies that our patients were, as a group, not as ill as those of other studies. Also, we observed that TNF- and IL-6, which can be useful markers of neonatal sepsis,^20,21,29,30 were only slightly elevated at study entry. Although GM-CSF, TNF-, and IL-6 did not differ after rG-CSF administration in our study, Görgen et al³¹ showed a reduction of mortality and suppression of TNF- activity when very high doses of G-CSF (250 µ/kg) were administered to rats with Gram-negative sepsis. Similarly, in rats with experimental sepsis, Lundblad et al³⁰ showed a reduction in mortality, bacterial count, TNF-, and endothelin-1 after rG-CSF treatment.

None of the previous reports of rG-CSF administration to infected neonates observed either short-term or long-term adverse effects. Similarly, we observed no adverse effect. Potentially, neutrophil-induced pulmonary injury could occur from rG-CSF administration, which might manifest itself as an increasing oxygen requirement or a longer time on mechanical ventilation.^32,33 We did not observe either. We did see an unexplained increase in blood monocyte concentration, similar to that reported by Gillan et al,¹⁵ Schibler et al,¹⁸ Kocherlakota and La Gamma,¹⁶ and Bedford-Russel et al,³⁴ but the thrombocytopenia described by Bedford-Russell et al³⁴ and not seen by Gillan et al,¹⁵ Schibler et al,¹⁸ or Kocherlakota and La Gamma¹⁶ was not seen in our patients.

	CONCLUSION

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The administration of rG-CSF to premature neonates who had the clinical diagnosis of early-onset sepsis resulted in no reduction in mortality but did result in a reduction in nosocomial infections over the subsequent 2 weeks. Additional studies are needed to confirm or refute our finding regarding prevention of nosocomial infections, and if the observation is validated, to elucidate the responsible mechanisms and to evaluate the issues of treatment benefits versus risks.

	ACKNOWLEDGMENTS

This study was supported by Grant 96063 from Fundo de Incentivo à Pesquisa, Grant 96114 from Grupo de Pesquisa e de Pós-Graduação do Hospital de Clínicas de Porto Alegre, and by Grants RR-00082, HL-44951, and HL-61798 from the National Institutes of Health.

	FOOTNOTES

Received for publication Nov 24, 1999; accepted Apr 17, 2000.

This work was presented in part at the Society for Pediatric Research; May 1-5, 1998; New Orleans, LA.

Reprint requests to (R.D.C.) Department of Pediatrics, University of Florida College of Medicine, 100296, Gainesville, FL 32610. E-mail: chrisrd{at}peds.ufl.edu

	ABBREVIATIONS

G-CSF, granulocyte colony-stimulating factor; r, recombinant; ANC, absolute neutrophil count; I/T, immature to total blood neutrophil; IL-6, interleukin 6; TNF-, tumor necrosis factor-; NEC, necrotizing enterocolitis; LC, leukocyte count; NPP, neutrophil proliferative pool; NSP, neutrophil storage pool; GM-CSF, granulocyte-macrophage colony-stimulating factor; SNAP, Score for Neonatal Acute Physiology.

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