PEDIATRICS Vol. 107 No. 1 January 2001, pp. 36-41

A Randomized Trial of Granulocyte-Macrophage Colony-Stimulating Factor in Neonates With Sepsis and Neutropenia

Kemal Blgn, MD, Ahmet Yarami, MD, Kenan Haspolat, MD, M. Ali Ta, MD, Sacit Günbey, MD, and Orhan Derman, MD

From the Department of Pediatrics, Dicle Medical School, Diyarbakr, Turkey.

	ABSTRACT

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Objectives.  To determine whether adjunctive therapy with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) could reverse sepsis-associated neonatal neutropenia and improve neonatal survival and to assess its safety compared with conventional therapy in a control group.

Study Design.  This prospective, randomized, controlled trial was performed in 60 infants with neutropenia and clinical signs of sepsis. A subcutaneous injection of rhGM-CSF (5 µg/kg/day) was administered to 30 of the patients for 7 consecutive days. Hematologic parameters (absolute neutrophil, eosinophil, monocyte, lymphocyte counts, and platelet number) and outcome were compared with 30 conventionally treated (control) patients.

Results.  Twenty-five patients from the GM-CSF-treated group and 24 from the conventionally treated group had early-onset sepsis (3 days' postnatal age), and the other 11 patients had late-onset sepsis (>3 days' postnatal age). There was no difference between groups in terms of birth weight; gestational age; gender; maturity; maternal age; and incidence of prolonged rupture of membranes, maternal hypertension, or severity of sepsis. All neonates tolerated GM-CSF well with no adverse reactions. The absolute neutrophil count on day 7 was significantly increased in the GM-CSF-treated group compared with the conventionally treated group: 8088 ± 2822/mm³ versus 2757 ± 823/mm³. The mean platelet count was significantly higher on days 14 in the GM-CSF-treated group compared with conventionally treated group: 266 867 ± 55 102/mm³ versus 229 200 ± 52 317/mm³. Hematologic parameters were otherwise similar between groups before treatment and on day 28. Twenty-seven neonates in the rh-GMCSF group and 21 in the control group survived to hospital discharge. The mortality rate in the rhGM-CSF group (10%) was significantly lower than in the conventionally treated group (30%).

Conclusion.  Treatment with rhGM-CSF is associated with an increase in absolute neutrophil, eosinophil, monocyte, lymphocyte, and platelet counts and decreased mortality in critically ill septic neutropenic neonates. These results suggest that rhGM-CSF may be effective in the treatment of neonatal sepsis with neutropenia, and further randomized trials are needed to confirm its beneficial effects.  Key words:  recombinant human granulocyte-macrophage colony-stimulating factor, neonates, neutropenia, sepsis.

Bacterial sepsis continues to be an important cause of morbidity and mortality in neonates. Four percent of deaths in the first 3 days of postnatal life and 45% of deaths after 2 weeks are related to infections, with no change in this pattern for the past 15 years.^1,2 Bacterial sepsis occurs in 1% to 10% of term newborns³ and is up to 50 times more common in infants with birth weight <800 g.⁴

Neonatal host defense is characterized by developmental immaturities in humoral, cellular, and phagocytic immunity.^5-8 Neutropenia in the neonate, as a result of decreased bone marrow neutrophil storage pool reserves and myeloid committed progenitor cells (granulocyte-macrophage colony-forming units), is also associated with an increased risk of morbidity and death during overwhelming bacterial sepsis.^9-15 Attempts to reduce sepsis-related mortality using intravenous immunoglobulin have failed to have a major impact,¹⁶ and attention has recently turned to the potential enhancement of phagocyte immunity using the hemopoietic colony-stimulating factors.¹⁷ Clinical trials in neonates have been preceded by extensive in vitro and animal studies because of concern about potential acute and long-term toxicity of such agents in the newborn period.¹⁷

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can prime term and preterm neutrophils for enhanced chemotaxis and respiratory burst responses,^18-20 and studies in newborn rats have demonstrated that both G-CSF²¹ and GM-CSF²² can increase survival after experimental sepsis, provided they are given before bacterial inoculation.²³ Thereafter, GM-CSF therapy in very low birth weight neonates (501-1500 g) was demonstrated to be safe and well-tolerated; recombinant human GM-CSF (rhGM-CSF) induced a significant increase in circulating and bone marrow neutrophils and, at high doses, increased the circulating monocyte and platelet counts.²⁴ In newborn with presumed sepsis, short-term treatment with G-CSF increased the neutrophil count²⁴ and, more importantly, improved survival.²⁵ GM-CSF has a wider spectrum of effect, stimulating the proliferation of both monocyte and neutrophil precursors²⁶ together with a greater effect on the bactericidal function of mature neutrophils than G-CSF,²⁷ as well as functionally enhancing monocytes.^28,29 The aim of this study was to determine whether rhGM-CSF might prove helpful in the treatment of neonatal sepsis-associated neutropenia in a randomized, controlled trial.

	METHODS

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Study Design and Patient Selection

This study was a prospective, randomized, controlled trial performed in the pediatric neonatal unit of the University Hospital of Dicle University at Diyarbakr, Turkey, from January 1994 to March 1995, with the approval of local institutional review boards and written parental consent. Relative risk (RR) of mortality was calculated (axd)/(bxc), where a, b, c, and d, respectively, are the numbers of marker (+) patients, marker () patients, marker (+) controls, and marker () controls. RR value <.5 was assigned as relatively low risk, and RR value >2 was assigned as relatively high risk.³⁰ Sepsis was diagnosed when a neonate with new symptoms (eg, worsening respiratory distress, new apnea, temperature instability, or other accepted clinical signs of sepsis²⁵) concurrently had at least one positive blood culture in the first 28 days of life. Blood cultures were repeated every 24 to 48 hours in all patients with a previous growth until the result was sterile. Neutropenia was defined as a total neutrophil count <1500 cell/mm³ using a minor but more stringent adaptation of the criteria of Manroe et al.³¹ All patients weighed between 1100 and 3500 g, had adequate renal and liver function, and had documented serum creatinine levels 2.0 mg/dL and serum glutamic-pyruvic transaminase 3 times normal. Exclusion criteria were the presence of major congenital anomaly or intrauterine infections. Infants were randomized, stratified by growth status as small for gestational age (SGA), large for gestational age (LGA), or appropriate for gestational age (AGA). SGA and LGA infants were defined as being below the 10th or above 90th percentile birth weight for gestational age, respectively. Patients were assigned alternatively to the rhGM-CSF or control groups according to their order of admission. Both groups (n = 30 each) were treated with appropriate conventional therapeutic interventions. Antibiotic regimens were modified during follow-up according to culture and antibiotic sensitivity tests, and patients whose blood culture were negative were excluded from the study. For infants in the study group, rhGM-CSF was given with the onset of signs of sepsis. The rhGM-CSF group received rhGM-CSF (Leucomax, Novartis, United Kingdom) at a dosage of 5 µg/kg/day subcutaneously via a 27 French gauge needle for 7 consecutive days. It was discontinued before 5 days if the total white blood cell (WBC) count exceeded 50 × 109/L. RhGM-CSF was supplied as lyophilized powder and reconstituted at a concentration of 5 µg in .1 mL. Infants were discharged when they were clinically recovered. Hematologic parameters on day 28 in these infants were obtained in an outpatient clinic.

Laboratory Evaluation

Complete blood counts were obtained by counter auto-analyzer machine at study entry and after treatment. WBCs were also counted on a hemocytometer, and differential leukocyte counts were performed manually on Wright-stained blood films every day. The absolute neutrophil count (ANC) was determined by the percentage of polymorphonuclear leukocytes (PMLs) and band forms identified manually. There was no significant difference with respect to results of ANC between the auto-analyzer machine and hemocytometer. The hematologic scoring system developed by Rodwell et al³² in 1993 was used for early diagnosis of neutropenic neonatal sepsis (Table 1). Metamyelocytes and myelocytes were not included because they are functionally inactive in the phagocytic defense pathway and contribute minimally to the total count.³³

Statistical Analysis

Comparisons between groups were made for weight on admission, demographic characteristics, change from baseline in hematologic responses, hospital stay, and mortality using Student's t test and ² tests.

	RESULTS

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Of the 60 infants, 16 were SGA, 2 were LGA, and the remainders were AGA. Thirty neonates were in the rhGM-CSF group and 30 in the control group. Twenty-five patients from the GM-CSF group and 24 from the conventionally treated group had early-onset sepsis (3 days' postnatal age) with a median age of onset of 1.3 days in the GM-CSF group and 1.5 days in the conventionally treated group. Five neonates from the GM-CSF group and 6 from the conventionally treated group had late-onset sepsis (>4 days' postnatal age) with a median age of onset of 12 days in both groups. The demographic and clinical characteristics of both groups showed no significant differences with regard to birth weight, gestational age, gender, maturity, maternal age, prolonged rupture of placental membranes (24 hours), and maternal hypertension (Table 2). The severity of illness was assessed by clinical criteria (eg, refusal of breastfeeding, crying, less spontaneous activity, cyanosis, irritability, dyspnea, hypothermia, apnea, sclerema, hypotonia, and cutis mormoratus), microorganisms isolated in blood culture, hematologic alterations (absolute neutrophil, absolute eosinophil, absolute monocyte, absolute lymphocyte, and mean platelet counts), and hematologic scoring at admission did not differ between groups.

Microorganisms isolated in blood culture in the rhGM-CSF group were Escherichia coli,²² coagulase-negative staphylococci,² Gram-negative bacilli,² group B streptococci,² Pseudomonas aeruginosa,¹ and Enterobacter sp.¹ Microorganisms in the conventionally treated group were E coli,¹⁷ coagulase-negative staphylococci,⁵ Gram-negative bacilli,⁴ group B streptococci,² P aeruginosa,¹ and Enterobacter sp.¹ Early-onset sepsis was caused by E coli, and late-onset sepsis by coagulase-negative staphylococci in both the rhGM-CSF and control groups. There was no significant difference between groups with respect to specific organisms. None of the patients had polymicrobial sepsis or recurrent sepsis, and none had positive follow-up blood cultures after the initial positive cultures.

All neonates tolerated rhGM-CSF well; no adverse reactions, such as skin rashes and irritability, were identified. Vital signs did not differ from baseline and electrolytes remained within normal limits.

Twenty-seven neonates in the GM-CSF-treated groups and 21 neonates in the conventionally treated groups survived to hospital discharge. There were significant differences in mortality rates between the GM-CSF-treated and the conventionally treated groups (P < .05). The occurrence of deaths related to sepsis was: 3 infants in the rhGM-CSF group (on days 7, 13, and 19 after onset of sepsis) and 9 in the control group (3 on day 5, and the others on days 6, 7, 9, 10, 11, and 12 after onset of sepsis). One neonate in the GM-CSF-treated groups and 3 neonates in the conventionally treated groups died from necrotizing enterocolitis (NEC). Infants with NEC diagnosed with clinically such as abdominal distention, bilious gastric residuals after feedings, vomiting, and/or gross or microscopic gastrointestinal bleeding. These patients died before the administration of the third dose of GM-CSF and in the first 4 days of conventional antibiotic treatment, respectively. Mortalities included 1 of the 25 early-onset sepsis neonates in the GM-CSF-treated groups and 3 of the early-onset sepsis neonates in the conventionally treated groups. When deaths attributable to NEC and deaths in late-onset sepsis (1 patient from each group) were excluded, significant differences in clinical recovery and mortality in favor of the rhGM-CSF group persisted (P < .01). Patients' mean duration of hospital stay from admission to discharge was 11 (5-19) days in the study group and 13 (5-21) days in the control group (P < .05). When patients who died are excluded from both groups, these figures are 11.1 ± 3.1 days (5-17 days) in the rhGM-CSF group and 15.3 ± 2.7 days (10-21 days) in the control groups (P < .01). The mortality risk in the GM-CSF-treated group and conventionally treated group were relatively 3.8 and .25, respectively. Treatment with rhGM-CSF in the neonates who had neutropenia and sepsis produced 15 times reduction in the RR of mortality.

Hematologic Responses

The ANC at the time of entry in the GM-CSF-treated group was 1100 ± 1342/mm³ and 1030 ± 1784/mm³ in the conventionally treated group. The ANC increased by threefold at 24 hours in the GM-CSF-treated group, while no change was noticed in the conventionally treated group. Subsequently, the ANC increased by fourfold versus twofold at 48 hours, fivefold versus twofold at 72 hours, and eightfold versus threefold at 7 days after entry (Fig 1A). In the GM-CSF-treated group, the number of infants no longer neutropenic on days 1, 2, and 3 were 28 (93.3%), 1 (1.3%), and 1 (1.3%), respectively. In the conventionally treated group, the number of infants no longer neutropenic on days 1, 2, 3, 4, and 5 were 3 (10%), 11 (36.6%), 12 (40%), 3 (4%), and 1(1.3%), respectively. The difference in neutrophil response became statistically significant on days 1, 2, and 3 of therapy between the 2 groups (P < .01).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Absolute neutrophil and monocyte at baseline and after rhGM-CSF (study group) and control group. For each treated group the pretreatment value is at day 0; the during-treatment value is the mean of counts obtained on study days 1 to 7; the 2-week posttreatment value is the mean of counts obtained on study day 14. Control participants were analyzed at equivalent time points. A, The ANC on days 1, 2, 3, 7, and 14 was significantly increased in the GM-CSF-treated group, compared with the conventionally treated group (P < .01. B, The absolute monocyte count was significantly higher on days 2, 3, 7, and 14 in the GM-CSF-treated group, compared with the conventionally treated group (P < .01).

The ANC on day 7 was significantly increased in the GM-CSF-treated group compared with the conventionally treated group: 8088 ± 2822/mm³ versus 2757 ± 823/mm³ (P < .01). The increase from baseline remained significantly elevated in the rhGM-CSF group on day 14, but was similar to the control group by day 28 of the study. rhGM-CSF was withheld in 9 patients (30%) because of leukocytosis.

The absolute eosinophil count was significantly higher on day 7 in the rhGM-CSF group compared with the control group: 84 ± 104/mm³ versus 17.7 ± 20.8/mm³ (P < .01); similarly the absolute monocyte count on day 7 (902.8 ± 533.5/mm³ vs 220.6 ± 145.2/mm³; P < .01; Fig 1B), the absolute lymphocyte count on day 7 (3519 ± 1413/mm³ vs 2350 ± 669/mm³; P < .01), and the mean platelet count on day 14 (266 867 ± 55 102/mm³ vs 229 200 ± 52 317/mm³; P < .01) were higher in the rhGM-CSF group. However, there was no significant difference between groups in the baseline and day 28 values of the ANC; absolute eosinophil, monocyte, and lymphocyte counts; and mean platelet count. The hematologic scores, similar before treatment (3.66 ± .66 in the rhGM-CSF and 3.40 ± .26 in the control group), became significantly different after treatment (.46 ± .93 vs 3.13 ± 1.45; P < .05).

	DISCUSSION

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Neutropenia is commonly associated with neonatal sepsis and worsens the prognosis.^10-13 The mechanism underlying postnatal neutropenia during the neonatal period seems to be a combination of a reduced whole-body neutrophil cell mass, together with reduced numbers of committed neutrophil progenitors in the bone marrow at birth and an inability to increase granulopoiesis in response to sepsis.^35,36

A recently published randomized study³⁷ using GM-CSF as a prophylactic measure to reduce nosocomial infections in very low birth weight neonates reported eosinophilia similar to that observed in our rhGM-CSF-treated neutropenic septic participants. GM-CSF has previously been described to increase eosinophils in vitro and in vivo.^38-41 The increased platelet count on day 14 observed in our study is in agreement with some previous reports⁴² but not others.⁴³ The 10% mortality in our rhGM-CSF group was lower than in control patients (30%; P < .05). Many questions remain over the most effective use of CSF in neonates. It is beyond dispute that the combination of sepsis and neutropenia is associated with high mortality.¹³ Thus, prevention of sepsis-induced neutropenia is likely to reduce mortality, and prevention of neutropenia during early postnatal life is likely to reduce the risk of acquiring a life-threatening infection.^31,45 Studies of therapeutic G-CSF are focused on whether mortality can be reduced when given to neonates with established sepsis and neutropenia. In one single-center study, 14 neutropenic neonates with systemic signs of infection who received G-CSF for 3 days had a reduced mortality, compared with similar, retrospectively selected controls.²⁵ In another randomized, placebo-controlled study involving a total of 20 infants with clinical signs of early-onset sepsis, G-CSF administered for 3 days did not alter disease severity or mortality.⁴⁴ GM-CSF affects both mortality rate and duration of hospital stay significantly in our study.

	CONCLUSION

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This study demonstrates that administration of rhGM-CSF in neutropenic septic neonates for 7 days seems to decrease the rate of mortality and duration of hospitalization and is apparently free of adverse effects. This treatment approach warrants further investigation in larger groups of patients with early- and late-onset neonatal sepsis with and without associated NEC. Although our study involves a large number of participants, further blinded, placebo-controlled investigations are needed to support our findings.

	ACKNOWLEDGMENTS

This study was designed and the clinical protocol written jointly by Drs Blgn, Yarami, Haspolat, Ta, and Günbey. Dr Derman recruited the participants and gathered data. Sr Yarami wrote the article.

We thank Banu Anlar, MD, Department of Pediatrics, University of Hacettepe, Ankara, for her critical reading of the manuscript and her skillful assistance. We also thank the neonatal nurses for their support and collaboration in this study.

	FOOTNOTES

Received for publication Aug 17, 1999; accepted Aug 9, 2000.

Reprint requests to (A.Y.) Dicle Üniversitesi Tp Fakültesi, Çocuk Sal ve Hastalklar A.B.D., Diyarbakr, Turkey. E-mail: dryaramis{at}hotmail.com

	ABBREVIATIONS

GM-CSF, granulocyte-macrophage colony-stimulating factor; rhGM-CSF, recombinant human granulocyte-macrophage colony-stimulating factor; RR, relative risk; SGA, small for gestational age; LGA, large for gestational age; AGA, appropriate for gestational age; WBC, white blood cell; ANC, absolute neutrophil count; PML, polymorphonuclear leukocyte; NEC, necrotizing enterocolitis.

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