Study locality and duration
This study was carried out at neonatology units of Mansoura University Children's Hospital and of Mansoura Insurance Hospital over one year. IRB approved the study and fully informed written consents were obtained from the neonates' parents or guardians before enrolment in the study. The study was registered in clinical trials database (clinicaltrials.gov, ID: NCT04474327).
An open-label, randomized controlled parallel intervention trial
The study was conducted on late preterm infants at a gestational age between 340/7 weeks and 366/7 weeks with a birth weight of more than 1.5 Kg with clinical evidence of neonatal sepsis. Neonatal sepsis was defined in the presence of one of the following signs; fever (> 38°C, rectal), hypothermia (< 36°C, rectal), tachycardia (heart rate (HR) > 180 beats/min) or bradycardia (HR < 100 beats/min), apnea, lethargy, feeding problems, mottled skin, convulsions, hypotonia plus at least 2 of the following laboratory findings; leukopenia (WBCs < 5,000 mm3), leukocytosis (WBCs > 20,000 mm3), thrombocytopenia (< 100,000 mm3), serum C-reactive protein (CRP) > 15 mg/L, fibrinogen > 150 mg/dL, or metabolic acidosis (base excess of <7 mmol/L); with (culture-confirmed) or without (culture-negative) a positive blood culture result [17; 18]. Infants presented initially with septic shock, multi-organ dysfunction syndrome (MODS), disseminated intravascular coagulopathy, or feeding intolerance requiring to be null per mouth or infants with major congenital malformations, chromosomal aberrations or postoperative patients were excluded from the study.
Infants were divided into Montelukast group and routine care group. Both groups received the same protocol of treatment of sepsis including antibiotics and supportive measures according to the policy of neonatal units and patients' needs. Infants in both groups were started on empirical antibiotics with Ampicillin at a dose of 50 mg /kg/12 hours combined with Aminoglycoside (Gentamycin) with a dose 4.5 mg/kg/36 hours for infants with suspected early-onset sepsis while for late-onset sepsis, Cloxacillin (50 mg/kg/dose) was used instead of Ampicillin. After that, antibiotics were changed as appropriate according to the results of culture and antibiotic sensitivity. The use of positive pressure ventilation and its duration, inotropic drugs usage and other supportive measures were guided and monitored by the treating physicians according to the policy of the neonatal units and patients' needs.
The Montelukast group received, in addition to the routine care, Montelukast sodium (Singulair, Merck sharp & Dohme Corp.) for ten days in a dose guided by the infant's birth weight as follow (infant's weight 1.5 to 2 kg received 1.5 mg once daily whereas greater than 2 kg received 2 mg). The given dose was calculated according to Kim and coauthors . Montelukast was given by oral route by dissolving one sachet containing four mg of the Montelukast sodium in four ml milk to get a concentration of 1 mg/1ml.
Infants were discharged from NICU if they fulfilled the following criteria: adequate oral feeding sufficient to support appropriate growth and gaining weight, the ability to maintain normal body temperature in a home environment and sufficient mature respiratory control that allow safe discharge in addition to acceptable bilirubin level and free of infection.
For policy of discontinuation of respiratory support; infants were weaned off mechanical ventilation if they fulfilled the following NICU guidelines: Spontaneous respiratory effort, Gag or cough with suctioning, Satisfactory blood gases (PH more than 7.25, Partial pressure of carbon dioxide (pCO2) less than 60 mmHg, and base deficit less than 8meq/L) on a mean airway pressure less than 8 cm H2O and frequency less than 30 breath /minute and saturation more than 88% on fraction of inspired oxygen (FiO2) less than 30% in the preceding 24 hours besides approval of the attending physician.
Infants were weaned off continuous positive airway pressure (CPAP) if they fulfilled the following NICU guidelines: Pressure of 3 to 6 cm H2O for 24 hours, FiO2 less than 30% to keep target saturation in the preceding 24 hours, respiratory rate less than 60 breath/minute, no single apnea requiring bagging in the preceding 24 hours, no more than 6 apneas requiring stimulation in the preceding 24 hours, satisfactory blood gases (PH > 7.25, pCO2 < 60 mmHg, and base deficit < 8meq/L) and infant tolerates time off CPAP during nursing care in addition to approval of attending physician.
Infants were weaned off oxygen therapy if they fulfilled the following NICU guidelines: Infant's saturation remained above 91% in less than 30% FiO2 for 24 to 48 hours or infant could tolerate a trial of discontinuing oxygen therapy to 21% FiO2 for 1 hour.
Primary outcome was serum TNF Alpha level, an inflammatory marker, at day 10 after receiving therapy. Serum TNF alpha level (TNFa ELISA kits, Sunred PeloBiotech GmbH, Germany) was measured by double-antibody sandwich ELISA technique . Secondary outcome measures included; needs and duration for invasive mechanical ventilation, needs and duration of non-invasive ventilation, needs and duration of inotropic support, the total duration of NICU admission, serum CRP level at day 10 after receiving therapy. Pediatric Logistic Organ Dysfunction (PELOD) score was performed by the treating physician on admission and at day 10 in order to follow improvement or deterioration of the studied patients in both groups. Furthermore, delta PELOD (score at day 10 – score on admission) was calculated. PELOD score is used in neonates to calculate sepsis-induced organ dysfunction . The score assesses cardiovascular function (heart rate and systolic blood pressure), neurologic function (Glasgow coma scale and pupillary reaction), hepatic function (AST and INR), pulmonary function (PaO2/FiO2, PaCO2 and whether the patient is on mechanical ventilation), hematologic function (WBCs and platelet count) and renal function (serum creatinine). Patient survival was recorded along the duration of NICU admission. In our research, adverse effects of Montelukast therapy such as diarrhea, vomiting, fever, cough, conjunctivitis and skin signs (rash, eczema, bruises and erythematous lesions) were observed .
Clinical data such as demographic data, cause of admission, site of infection (bacteremia, pneumonia, meningitis or septic arthritis), vital signs, activity, feeding tolerance, respiratory symptoms, duration of respiratory support, use of inotropes and a venous sample was withdrawn for complete blood picture, serum CRP, TNF alpha, creatinine, liver enzymes, INR and blood culture. Blood sampling in the studied groups was performed twice: First, at the start of the study, once sepsis was suspected, and the second sample was after 10 days of treatment. Lumbar puncture was performed when meningitis was suspected.
Randomization and allocation
Infants were assigned randomly to treatment groups using internet-based random table technique with a block size of four. Cards in sequentially numbered, opaque, sealed envelopes kept in the NICU. A designated nurse who was not involved in the study was responsible for the randomization of selected infants.
Sample size calculation
Sample size calculation was based on mean TNF level between the studied groups (Routine care group & Montelukast group). Pilot study was carried out on 10 cases (5 in each group) to calculate sample size. Using G power calculator to calculate the difference between 2 means using t-test with an effect size of 0.894, 2-tailed, with α error = 0.05 and power = 80%, mean ± SD of mean TNF level (110±30 and 90.73±10), the total calculated sample size was 40 infants (20 in each group).
IBM’s SPSS statistics (Statistical Package for the Social Sciences) for Windows, version 20 was used for statistical analysis of the collected data. Shapiro-Wilk test was used to check the normality of the data distribution. Quantitative variables were expressed as mean + standard deviation or median (minimum-maximum) as appropriate while categorical variables were expressed as frequency and percentage. Independent sample t-test and Mann Whitney tests were used for inter-group comparison of parametric and non-parametric continuous data. Chi-square /Fisher´s Exact tests were used for inter-group comparison of nominal data using the crosstabs function. P (probability) value < 0.05 was considered statistically significant.