Sepsis-Related Mortality of Very Low Birth Weight Brazilian Infants: The Role of Pseudomonas aeruginosa

The aim of this study is to identify risk factors for sepsis-related mortality in low birth weight (<1500 g) infants. We performed retrospective cohort study to investigate risk factors for sepsis-related mortality in all neonates birth weight <1500 g admitted to Level III neonatal intensive care unit, Brazil, April 2001/September 2004. Of the 203 cases, 71 (35%) had sepsis. Of those, gram-positive was identified in 52/87 blood cultures (59.8%), the most common Coagulase-negative Staphylococcus (31/87; 35.5%). Gram-negative was present in 29 of the 87 positive blood cultures (33.3%), with Pseudomonas aeruginosa (8/87; 9.1%), the most frequent agent. Overall 21 of 71 infants with sepsis (29.6%) died. Risk factors for sepsis-related mortality were gestational age ≤28 weeks, birth weight ≤1000 g (9.6 times more often than birth weight >1000 g), five-minute Apgar ≤7, gram-negative sepsis, mechanical ventilation (6.7 times higher than no use), and intravascular catheter. Sepsis-related mortality was due, mainly, to Pseudomonas aeruginosa; birth weight ≤1000 g and mechanical ventilation were strong sepsis-related mortality predictors.


Introduction
Neonatal sepsis is a frequent complication of very low birth weight (VLBW) infants and it is an important cause of neonatal morbidity and mortality [1,2]. VLBW infants develop 2.7 times more sepsis than other infants since their immune system and skin barrier are immature and they are exposed to many invasive diagnostic and therapeutic procedures [3]. Morbidity and mortality in a neonatal intensive care unit (NICU) can be reduced by knowledge of the epidemiology of the microbiology, infection rate profile and antibiotic sensitivity, and by introducing practices that are based on clinical evidence [4,5]. Special attention must be given to infection by Pseudomonas aeruginosa due to a high mortality rate [6]. The aim of this study is to identify the risk factors involved in the mortality caused by sepsis of a VLBW infants population with sepsis, in particular, the role of Pseudomonas aeruginosa, a very aggressive pathogen. The infants were hospitalized in the NICU of a high-risk maternity in a tertiary-level public hospital specialized in caring for highly complex patients. Secondary objectives are to determine the frequency and distribution of the pathogens 2 International Journal of Pediatrics that cause infection and to describe the characteristics of this population.

Patients.
All VLBW infants were born at the maternity of Servidores do Estado Hospital (SHE), Rio de Janeiro, RJ, Brazil and admitted in the NICU of the same hospital, just after birth, between 1 April, 2001 and 30 September, 2004 meeting the following criteria: birth weight (BW) <1500 g, clinical evidence of systemic infection and positive blood culture result for bacterium or yeast on one or more blood cultures obtained at any time while infants were inpatients in the NICU. Infants died of nonsepsis causes, with lethal congenital malformations and chromosomal abnormalities were excluded.

Chart
Review. This retrospective cohort study collected data from the medical records of the selected VLBW infants using an investigation protocol. Relevant informations included Antenatal and Intrapartum History: gestational age (GA) based on the date of the last menstruation, uterus fundus measurement, and ultrasonography performed until 12 weeks of GA; time elapsed between rupture of membranes and birth; mode of birth; Apgar score at the first, fifth, and tenth minute of life; gender; BW; BW and GA relationship according to Usher and McLean's growth curves [7]. Events Related to Infection Episodes: incidence of sepsis, defined by the presence of clinical signs and symptoms as apnea, gastrointestinal problems, increased need for oxygen or ventilatory support, and lethargy/hypotonic, and by one or more positive blood cultures for bacterium or yeast obtained at any time during the infant's stay at the NICU. Any organism, including Coagulase-negative Staphylococcus (CONS) was considered the caused agent of sepsis and not a contaminant if the criteria of the Vermont Oxford Network Database were present: clinical signs of sepsis, positive blood culture for CONS, and intravenous antibacterial therapy for at least five days after obtaining blood culture or until death, in case it occurs within five days after obtaining blood culture [8]. Whenever CONS and another pathogen were identified in the same blood culture, only the other pathogen was recorded in the database, CONS was considered contaminant and this agent was discharged; age at the time of onset of sepsis considered to be the day on which the first blood culture for the event was positive: early-onset sepsis (EOS) defined as infection occurring before or at 72 hours of life and late-onset sepsis (LOS), after 72 hours of life [9]; organisms cultured and associated with death from sepsis; Neonatal Comorbidities: perinatal asphyxia defined as the presence of five-minute Apgar score below six and signs of encephalopathy as lethargy/stupor, hypotonia, and abnormal reflexes [10]; respiratory distress syndrome (RDS) defined as the need for oxigenotherapy, clinical features of RDS, and ventilation support and abnormal thorax radiograph in the first 24 hours of life [3]; necrotizing enterocolitis (NEC) classified according to the system of Bell et al. [11]; temperature instability, defined as temperature <36.5 • C or >37.5 • C or a variation of >1 • C in a period of 24 hours and blood changes such as neutropenia, defined by a neutrophil count < 1.5 × 10 3 /L and thrombocytopenia, defined as a platelet count < 80 × 10 3 /L [11]. Therapeutic Interventions: use and length of use of intravascular catheter, defined as a peripherally inserted central catheter or by a vascular dissection and arterial or venous umbilical catheter, inserted before the onset of sepsis; use and length of use of mechanical ventilation and of total parenteral nutrition (TPN); time when enteral feeding was initiated and length of stay in the NICU. Clinical Outcome: neonatal death was considered related to infection when clinical signs and symptoms of sepsis evolved to signs and symptoms of irreversible septic shock as systemic hypotension, acidemia, anuria, and hypoxia; and/or signs and symptoms of irreversible disseminated intravascular coagulation syndrome as thrombocytopenia, major bleeding episodes as pulmonary hemorrhage, and, ultimately, progressing to death.

Statistical Analysis.
Bivariate analysis was used to evaluate the association between potential risk factors and sepsisrelated mortality. For statistical inference, the proportions were compared with the chi-square test (χ 2 ) or with Fisher's exact test with Yates correction when the expected value in any cell of a 2 × 2 table is <5. P-value of .05 or less was considered significant. The cut-off for continuous data was obtained with the Receiver Operator Characteristics (ROC) curve. The cut-off was determined for the value with the highest accuracy from the area of the ROC curve, it means, to the highest area under the ROC curve. The odd ratios (ORs) were calculated with confidence intervals of 95% (CI95) for each risk factor. A stepwise backward unconditional multivariate logistic regression was performed to control for confounding and to assess the independence of the identified risk factors. All variables with a P-value of .05 or less on bivariate analysis were included in the initial model. ORs and CI95 were calculated. The final model included the statistically significant variables in unconditional logistic regression analyzed by P-value of the test Walds. Statistical analysis was performed using the software Epi Info version 3.3.2, SPSS version 12, and Epi Data version 3.1.
The study was reviewed and approved by the local Research Ethics Committee (no. 000197).  There were significant differences in death rates from sepsis, depending on the organisms isolated from the last positive culture ( Table 3). Episodes of sepsis with gramnegative organisms were more likely to result in death than episodes with gram-positive or fungus (13/29; 44.8% versus 7/52; 13.4% versus 1/6; 16.7%; P < .05). Within this highrisk group, VLBW infants with P. aeruginosa sepsis were most likely to die (6/8; 75% of infants with P. aeruginosa infections died; P < .05).
VLBW infants submitted to intravascular catheter and mechanical ventilation were significantly more likely to die than those not submitted to these procedures (19/47; 40.4% versus 2/24; 8.3%; and 19/42; 45.26% versus 2/29; 6.9%; P < .05 each one). Enteral feeding onset before or at 72 hours of life and length of stay were highly significant predictors of survival (P < .05). The bivariate analysis is shown in Table 4.

Logistic
Regression. The predictive model based on a stepwise backward unconditional multivariate logistic regression for 71 VLBW infants showed that BW independently contributed most to the dependent variable death, with a cut-off point of 1000 g. BW ≤1000 g infants presented a sepsis-related mortality rate 9.6 times higher than BW > 1000 g infants (P < .05). The use of mechanical ventilation presented a clinically significant risk (sepsis-related mortality rate 6.7 times higher) but the statistical significance was marginal (P = .05). Length of stay was statistically significant with a negative coefficient, that is, the risk of death from sepsis decreased as length of stay increased (P < .05). The logistic regression analysis is shown in Table 5.

Discussion
In our four-year cohort, sepsis was frequent, 35%, with the highest risk of occurrence in the first two weeks of stay. Even thought was caused mainly by gram-positive organisms, represented by CONS, the mortality was higher when caused by gram-negative organisms, particularly P. aeruginosa. Neonatal sepsis-related mortality rate was high, 29.6%, in most cases involving ELBW infants, 56.7%, and due to gramnegative organisms, 44.8%, especially P. aeruginosa, 75%. The number of VLBW infants studied was low, however, representative of the whole population of neonates with BW < 1500 g assisted in a NICU of a reference center for high-risk pregnant women, during the selected period. Being a retrospective study, there was a lack of comprehensive information about risk factors. For this reason, the possible risk factors of sepsis-related mortality were reported under Chart Review and included characteristics of the VLBW infants population and events related to infection episodes.
There was a predominance of gram-positive organisms in EOS, especially CONS. This finding differs from the reports of Stoll et al. [9] and Rønnestad et al. [12] that, in the recent years, there was a change in the profile of the pathogens causing EOS in NICU, with increase of gramnegative organisms, especially Escherichia coli. The incidence of EOS due to Group B Streptococcus (GBS) was low, a single episode, probably because, in most cases the evaluated VLBW infants were product of the interruption of a high-risk pregnancy by caesarian section with ruptured of amniotic membranes at birth. According to Hickman et al. [13], these two factors contribute to the decrease of the vertical transmission of GBS and the lowest occurrence of EOS due to this agent, as in our study.
There was a predominance of gram-positive organisms in LOS, 48.2%, mainly CONS, 27.5%. Costa et al. [14] in Portugal, Khashu [15] in Canada, Sarkar et al. [16] in the United State of America, and Richards et al. [17] in Colombia reported similar findings, what demonstrates that these organisms are prevalent agents of LOS in different countries and continents. P. aeruginosa was the most frequent agent of LOS, 9.1%, among gram-negative organisms. In contrast to our findings, Afroza [1] and Trotman and Bell [5] reported K. pneumoniae as the most usual infectious agent among gram-negative organisms in LOS. The reason for the high incidence of P. aeruginosa in our population must be object of future studies.
The sepsis-related mortality rate of VLBW infants was high, 29.6%, when compared to the rates reported in the literature, 17.3% to 21% [5,6,17]. VLBW infants with gram-negative sepsis were at the greatest risk of death, 44.8%. P. aeruginosa was the most aggressive agent of sepsis, responsible for the highest sepsis-related mortality rate, 75%, as reported by studies of Gordon and Isaacs [6].
BW ≤ 1000 g was identified by logistic regression as the strongest independent sepsis-related mortality predictor. ELBW infants, a great vulnerable population to neonatal complications, presented the highest sepsis-related mortality rate, 56.6%. Afroza [1] related that, among many risk factors for infection, the single most important factor is low birth weight, as we observed. This finding suggests that efforts to reduce neonatal sepsis-related mortality must be turned especially to this population.
The use of mechanical ventilation was identified by logistic regression as a strong independent sepsis-related mortality predictor, which is in agreement with Flidel-Rimon et al. [18] and Stoll et al. [19]. This may be caused by the fact that neonates who need ventilation are the sickest patients, but also may be in part related to preventable ventilatorassociated infection.
Enteral feeding initiated before or at 72 hours of life was a predictor of survival. Our findings suggest the practice of early onset of feeding, and of the discerning indication and maintenance of invasive procedures directed to the treatment of VLBW infants to control sepsis-related mortality in this population.
In summary, sepsis-related mortality is very high in low birth weight infants, mainly in extreme low birth infants. This is particularly true for patients with positive blood   Odds ratio (OR) for the probability of death by sepsis IC95 = 95% confidence interval.
(a) variable used in step 1 length of stay >28 days (b) variable used in step 2 birth weight ≤1000 g.

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International Journal of Pediatrics cultures due to P. aeruginosa. Attention to the control of prematurity, knowledge of the neonatal flora and antibiotic sensitivity profile, introduction of early nutritional support with early onset enteral feeding and efforts to decrease ventilator related events may play a role in decreasing the mortality and morbidity of this very serious disease.