|Year : 2018 | Volume
| Issue : 3 | Page : 970-976
Serum polymorphonuclear leukocyte elastase enzyme level in neonatal sepsis
Ahmed T Mahmoud1, Dalia M El-Lahouny1, Khalid A Khalifa2, Mohamed R Hosny3
1 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Pediatrics, Qwuesna General Hospital, Menoufia, Egypt
|Date of Submission||07-Jan-2017|
|Date of Acceptance||19-Mar-2017|
|Date of Web Publication||31-Dec-2018|
Mohamed R Hosny
Source of Support: None, Conflict of Interest: None
The aim of this study is to evaluate the clinical importance of polymorphonuclear leukocyte (PMN) elastase level in neonatal sepsis.
Neonatal sepsis is defined as a clinical syndrome of bacteremia with systemic signs and symptoms of infection in the first 4 weeks of life. When pathogenic bacteria gain access into the bloodstream, they may cause overwhelming infection without much localization (septicemia) or may be predominantly localized to the lung (pneumonia) or the meninges.
Patients and methods
This prospective study was performed on 60 newborns, which included 30 newborns with positive clinical sepsis score and 30 healthy neonates matched for age, sex, and weight as a control group. Blood samples for blood culture, routine biochemistry, whole blood count, immature neutrophil: total neutrophil ratio, C-reactive protein, and PMN elastase were taken. Antibiotics were commenced after the blood specimens were collected. Second blood samples for sepsis markers were obtained from the patient only on the fourth day of treatment.
In our study, we found that respiratory distress was the most common clinical presentation (86.7%) in neonates with sepsis followed by poor activity (83.3%) and jaundice (40%). PMN elastase levels in neonates with sepsis were highly significantly increased compared with controls. Overall, 100% of patients had a positive PMN elastase test result, and PMN elastase has a sensitivity of 96% and positive predictive value of 86% in prediction of sepsis. The sensitivity of C-reactive protein in detecting sepsis was 77%, specificity was 93%, positive predictive value was 87%, and negative predictive value was 14%.
These findings indicate that PMN elastase level is a major indicator for the early diagnosis of sepsis in newborns.
Keywords: C-reactive protein, early diagnosis, polymorphonuclear leukocyte elastase, sepsis
|How to cite this article:|
Mahmoud AT, El-Lahouny DM, Khalifa KA, Hosny MR. Serum polymorphonuclear leukocyte elastase enzyme level in neonatal sepsis. Menoufia Med J 2018;31:970-6
|How to cite this URL:|
Mahmoud AT, El-Lahouny DM, Khalifa KA, Hosny MR. Serum polymorphonuclear leukocyte elastase enzyme level in neonatal sepsis. Menoufia Med J [serial online] 2018 [cited 2019 Jun 16];31:970-6. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/970/248738
| Introduction|| |
Neonatal sepsis is defined as a clinical syndrome of bacteremia with systemic signs and symptoms of infection in the first 4 weeks of life. When pathogenic bacteria gain access into the bloodstream, they may cause overwhelming infection without much localization (septicemia) or may be predominantly localized to the lung (pneumonia) or the meninges (meningitis). Sepsis encompasses various systemic infections of the newborn such as septicemia, meningitis, pneumonia, arthritis, osteomyelitis, and urinary tract infections (UTIs). Superficial infections like conjunctivitis and oral thrush are not usually included under neonatal sepsis. The WHO estimates that one million deaths per year (10% of all under-five mortality) are because of neonatal sepsis and that 42% of these deaths occur in the first week of life.
The mortality rate in neonatal sepsis may be as high as 50% for infants who are not treated. Infection is a major cause of fatality during the first month of life, contributing to 13–15% of all neonatal deaths. Neonatal meningitis, a serious morbidity of neonatal sepsis, occurs in two to four cases per 10 000 live births and significantly contributes to the mortality rate in neonatal sepsis; it is responsible for 4% of all neonatal deaths. Term male infants have two-fold higher incidence of sepsis than term female infants, and this sex distribution is less clear in preterm low-birth-weight infants. Another study done by Moor and Badrawi has attributed rates of sepsis exceeding 50% in a neonatal ICU.
The earliest signs of sepsis are often subtle and nonspecific; indeed, a high index of suspicion is needed for early diagnosis. Signs of respiratory distress, including tachypnea, grunting, flaring of the nose, retractions, and decreased breath sounds are common and important findings in the infant suspected of having sepsis. Apnea is one of the most specific signs of sepsis but usually occur late.
The evaluation of tests for neonatal sepsis is important because the infection may present a very serious threat to the baby. It is urgent to know whether the baby has sepsis to start treatment as quickly as possible.
Polymorphonuclear leukocyte (PMN) elastase is one of the serine proteases found in the azurophilic granules of neutrophils. It is capable by itself of degrading several different structural components such as collagen, proteoglycan, heparin, and cross-linked fibrin. The activation of PMN leads to the release of multiple microbicidal products, including reactive oxygen species, cationic peptides, eicosanoids, and proteolytic enzymes.
There are numerous studies indicating that neutrophils play an important role in the pathogenesis of neonatal sepsis and multiple organ failure. Various published studies have shown PMN elastase to be a useful marker of early infection in the newborn. Tsaka and Herkner showed that newborns with sepsis had significantly increased PMN elastase levels at the time of recognition of infection. Wojsyk-Banaszak and Szczapa and Jensen et al. found that PMN elastase is higher in newborns with sepsis than those without sepsis. As bacterial infection leads to production, activation, and damage of PMNs, one would expect the extent of granulocyte elastase release to parallel the severity of infection.
The aim of the study was to determine the role of PMN elastase as diagnostic marker in neonatal sepsis.
| Patients and Methods|| |
This prospective study was conducted on 30 neonates from neonatal ICU, and 30 healthy neonates from Pediatric Outpatient Clinic for routine follow-up at Menoufia University Hospital in the period from September 2015 to July 2016.
They were divided into two groups:
Group 1 (patients group) included 30 neonates with positive clinical sepsis score. The study was performed on the first day of suspected sepsis (pretreatment) and on the fourth day after treatment with antibiotics of the same patients (post-treatment).
Group 2 (control group) included 30 healthy neonates matched for age and sex with the patient group.
The study was approved by Ethical Committee of Menoufia Faculty of Medicine. An informed consent was taken from parents of each neonate included in this study.
The inclusion criteria were neonates with signs and symptoms suggestive of neonatal sepsis according to Tollner score.
Neonates with either congenital infection, suspected inborn error of metabolism, perinatal asphyxia, congenital anomalies, or chromosomal abnormalities; preterm neonate; and those with low birth weight were excluded from the study.
A total of 30 newborns with positive clinical sepsis score were included. The control group included 30 healthy neonates matched for age, sex, and weight.
All laboratory tests were done initially (first day of suspected sepsis) and repeated on the fourth day of antibiotics treatment only for cases, whereas for the control group, laboratory tests were done only once.
Blood samples for blood culture, routine biochemistry, whole blood count, immature neutrophil: total neutrophil (I/T) ratio, C-reactive protein (CRP), and PMN elastase were taken.
- Complete blood picture: it was performed using Udihem-I No: 803535 blood cell counter (United Diagnostic Company, 1440 Taft Avenue, 1000 Manila, Philippines)
- CRP was determined using Avitex-CRP kit (Omega Diagnostics, Omega House, Hillfoots Business Village, Alva, FK12 5DQ Scotland, United Kingdom), which is a rapid latex agglutination test kit
- Blood culture: in neonatal bottles, blood was collected and subcultured on blood agar plate. When a blood culture tested positive within 72 h, it was considered as a case of true bacteremia. Aerobic and anaerobic culture preparations on blood agar plates at 10% CO2 and on MacConkey agar were done. Isolated colonies were identified by colony morphology, Gram smears, and biochemical and enzymatic reactions. If no growth was obtained, the bottles were incubated up to 10 days, with subculture every other day on solid media. If no growth occurred after 10 days of incubation, blood culture was considered negative
- Serum PMN elastase level was analyzed by specific enzyme-linked immunosorbent assay kit (Immundiagnostik, Bensheim, Germany) according to the manufacturer's instructions.
Results were collected, tabulated, and statistically analyzed using IBM personal computer and statistical package SPSS version 20 (SPSS Inc., Chicago, Illinois, USA). Two types of statistics were done: descriptive statistics included percentage (%), mean (x), and SD.
Student's t-test is a test of significance used for comparison between two groups having quantitative variables, and Mann–Whitney U-test is a test of significance used for comparison between two groups having quantitative variables not normally distributed. ROC curves were used to compare the diagnostic performance of two or more laboratory or diagnostic tests. P values less than 0.05 was considered statistically significant.
| Results|| |
There were no statistically significant differences between neonates with sepsis and controls regarding sex, mode of delivery, gestational age, birth weight, and postnatal age, as shown in [Table 1]. In our study, we found that that premature rapture of membrane (PROM) was the highest maternal risk factor among patient group (36.7%), followed by maternal fever (26.7%), maternal UTI (13.3%), pre-eclampsia (3.3%), and maternal diabetes (3.3%). Approximately 16.7% of the patients with sepsis had no history of maternal risk factor, as shown in [Table 2]. In our study, respiratory distress was the most common clinical presentation (86.7%) in neonates with sepsis followed by poor activity (83.3%) and jaundice (40%), as shown [Figure 1]. We found that that hemoglobin level and platelet count in neonates with sepsis were highly significantly decreased compared with the control group. On the contrary, I/T ratio, total leukocyte count, and CRP were highly significantly increased in patients with sepsis than controls, as shown in [Table 3].
|Table 2: Frequency distribution of maternal risk factors among neonatal patients with sepsis|
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|Figure 1: Frequency distribution of clinical presentations among neonatal patients with sepsis.|
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|Table 3: Complete blood picture, C-reactive protein, and polymorphonuclear leukocyte elastase (ng/ml) in neonatal patients with sepsis and controls|
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In our study, we found that PMN elastase levels in neonates with sepsis were highly significantly increased compared with controls, as shown in [Table 3]. We found that 96.7% of the patients had a positive blood culture and 4% had a negative blood culture; moreover, 100% of patients had a positive PMN elastase test result, and 76.7% of patients had a positive CRP as shown in [Table 4].
|Table 4: Percentage of the positive and negative blood cultures, C-reactive protein, and polymorphonuclear leukocyte elastase in the patient group|
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Regarding blood culture, the most common organisms in neonates with sepsis were Gram-negative organisms (66.6%) followed by Gram-positive organisms (16.7%), and the least common is fungal infection (3.3%). Klebsiella pneumoniae was the most common organism (33.3%) isolated from blood cultures followed by Pseudomonas aeruginosa (16.7%) and Staphylococcus aureus (16.7%), Escherichia coli (13.3%), Enterobacter spp. (3.3%), and Candida albicans (3.3%), as shown in [Figure 2]. As shown in [Table 5], the highest specificity (100%), sensitivity (96%), positive predictive value (PPV) (86%), and negative predictive value (NPV) (90.9%) were found for PMN elastase levels in prediction of sepsis. The sensitivity of CRP in detecting sepsis was 77% and its specificity was 93%.
|Table 5: Specificity and sensitivity of polymorphonuclear leukocyte elastase, C-reactive protein, total leukocyte count, platelet count, and immature neutrophils/total neutrophils in early diagnosis of sepsis|
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In our study, we found that there were highly significant lower levels of PMN elastase, CRP, TLC, and I/T after treatment in neonates with sepsis. Otherwise, there were highly significant higher levels of hemoglobin and platelets after treatment, as shown in [Table 6]. As shown in [Table 7], it was found that there were significant positive correlation between PMN elastase and TLC, I/T ratio, and CRP and significant negative correlation with hemoglobin level and platelet count.
|Table 6: Laboratory markers levels before and after treatment in neonatal patients with sepsis|
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|Table 7: Correlations between polymorphonuclear leukocyte elastase levels before treatment with the studied variables in neonatal patients with sepsis|
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| Discussion|| |
The WHO estimates that one million deaths per year (10% of all under-five year mortality) are because of neonatal sepsis and that 42% of these deaths occur in the first week of life. Early treatment of neonatal sepsis is vital to improve outcome. In the absence of reliable infection markers during the first hours of life, neonatologists often begin early antibiotic treatment in newborn infants with risk factors for infection, exposing many neonates to unnecessary treatment. Considering the high mortality and serious morbidity among neonates experiencing sepsis, a diagnostic marker with high sensitivity and specificity is desirable. However, there is no laboratory marker that has all the characteristics of an ideal infection marker.
In our study, there was no significant difference in patients and control group regarding sex. This was in agreement with Betty and Inderpreet, who did not observe any increase in incidence of sepsis among the studied male neonates. However, Albers et al. stated that male infants have a fourfold increased risk to develop sepsis than females; this may be owing to the presence of gene located on X chromosome which is involved in the function of the thymus or with synthesis of immunoglobulin.
In the current study, analysis of maternal risk factors for sepsis revealed that PROM was the highest maternal risk factor among patient group (36.7%) followed by maternal fever (26.7%), maternal UTI (13.3%), pre-eclampsia (3.3%), and maternal diabetes (3.3%). In the studies done by Lopez-Sastre et al. and Ramasethu, it was found that chorioamnionitis was a significant risk factor for neonatal sepsis. Moreover, El-Mashad et al. approved that PROM more than 18 h and maternal UTI were significant risk factors for neonatal sepsis. On the contrary, Kaufman and Fairchild and Ottolini et al. found that maternal UTI was a major maternal risk factor for neonatal sepsis followed by PROM more than 18 h and maternal fever.
Regarding the clinical presentation among septicemic group, respiratory distress was the most common clinical presentation (86.7%) in neonatal patients with sepsis followed by poor activity (83.3%) and jaundice (40%). As unlikely observations, Bizzarro et al. found that the presenting signs and symptoms at onset of infection in late-onset sepsis were hypothermia (temperature <36.5°C) (41%), apnea (38.2%), blood glucose more than 140 mg/dl (38.3%), bradycardia (30.3%), fever (temperature >38°C) (21.6%), and blood glucose less than 40 mg/dl (6.9%). Therefore, clinical signs and symptoms of sepsis are nonspecific, and the differential diagnosis is broad, including most if not all body systems because neonatal infection may be limited to a single organ or may involve multiple organs.
In the current study, it was found that positive blood cultures were 96.6% in the sepsis group. Chacko and Sohi found that culture-proven sepsis was found in 41.6% of cases with sepsis.
In this study, the most common organisms isolated from septic neonates were Gram-negative organisms (66.6%) followed by Gram-positive organisms (16.7%), and the least common organisms is fungal infection (3.3%). K. pneumoniae was the most common organism (33.3%) isolated from blood cultures followed by P. aeruginosa (16.7%) and S. aureus.
Bizzarro et al. stated that the predominant organism cultured was coagulase-negative Staphylococci followed by E. coli, C. albicans, Gram-negative rods other than E. coli, and commensal species.
In our study, it was detected that PMN elastase was positive in 100% of the patients. Moreover, we found in our study marked reduction in level of PMN elastase in the serum of the patient group after treatment.
In this study, it was detected that PMN elastase levels of newborns with sepsis were significantly higher than controls (P < 0.001). This agrees with Tsaka and Herkner who observed that newborns with sepsis had significantly increased PMN elastase levels at the time of recognition of infection. Wojsyk-Banaszak et al. and Jensen et al. found that PMN elastase is higher in newborns with sepsis than in newborns without sepsis. Laskowska et al. also found that in full-term neonates, cord blood neutrophil elastase is a good marker of infection. In our study, there was no significant difference in PMN elastase between culture-positive and culture-negative patients.
In our study, there was a positive correlation of PMN elastase with TLC and I/T ratio and negative correlation with platelet count and hemoglobin level.
In our study, the sensitivity of PMN elastase in the diagnosis of neonatal sepsis was 96%, specificity was 100%, PPV was 86%, and NPV was 90.9%. Jensen et al. reported that the sensitivity and a specificity of serum PMN elastase in the early diagnosis of neonatal sepsis were 76 and 81%, respectively. In our study, we demonstrated that CRP had a moderate sensitivity (77%) and high specificity (93%) for detecting neonatal sepsis. This is in agreement with the study by Caldas et al., in which CRP has a moderate sensitivity (78.6%) and high specificity (87.5%) for detecting neonatal sepsis.
The results of this study showed that the sensitivity, specificity, PPV, and NPV of the PMN elastase were high for early diagnosis of neonatal sepsis. Our findings suggest that PMN elastase is an almost perfect marker and more sensitive and specific than all other parameters in the diagnosis of neonatal sepsis. However, lack of correction for reference ranges for neonatal PMN elastase values may influence the outcome of PMN elastase as a marker for bacterial infection. In addition, methodological difficulties in detecting PMN elastase and the absence of their routine usage in all centers have limited its use in daily practice.
| Conclusion|| |
We found that PMN elastase is an important marker in the diagnosis of neonatal sepsis, and this marker is also valuable in following the effectiveness of treatment and determining the prognosis of the disease when used with CRP and clinical signs.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]