Platelet to Lymphocyte Ratio and Neutrophil to Lymphocyte Ratio as New Diagnostic Markers for Detection of Early-onset Neonatal Sepsis in Full-term Newborns.

Objective: The purpose of this study was to investigate the clinical signicance of the platelet to lymphocyte ratio (PLR) and the neutrophil to lymphocyte ratio (NLR) in term neonates and its impact on management of Early-Onset Neonatal Sepsis (EOS). Materials and Methods: This prospective cross-sectional observational study was conducted with 40 term neonates diagnosed with EOS compared with 40 healthy controls. Exclusion criteria were prematurity, post- maturity, small or large for gestational age according to week of pregnancy, preeclampsia, gestational diabetes mellitus, chorioamnionitis, congenital major anomalies, and cyanotic congenital heart disease. Results: A total of 80 term neonates were included in the study. Of these, 40 were diagnosed with EOS and 40 were healthy controls. NLR and PLR as predictors of early-onset neonatal sepsis, sensitivity of NLR was 67% and PLR was 70% and specicity of NLR was 99% and PLR was 73% and PPV of NLR was 98%, PLR was 72%. There is a signicant weak positive correlation between platelets and sepsis, signicant fair positive correlation between WBCs and PLR with sepsis, signicant moderate positive correlation between immature neutrophils, I.T and NLR with sepsis, nally a signicant negative fair correlation between lymphocytes and sepsis. Conclusions: NLRs and PLRs were positively correlated with EOS in term neonates, and these ratios can be used as diagnostic adjunct tests for neonate EOS workups.


Introduction
Neonatal Sepsis is an important cause of morbidity and mortality among newborn infants. Although the incidence of sepsis in term and late preterm infants is low, the potential for serious adverse outcomes is of such great consequence that caregivers should have a low threshold for evaluation and treatment for possible sepsis in neonates (1).
Neonatal sepsis a systemic condition of bacterial, viral, or fungal (yeast) origin that is associated with hemodynamic changes in an infant 28 days of life or younger, the de nition of sepsis has included isolation of a pathogen from a normally sterile body uid such as blood or cerebrospinal uid (CSF). However, as the clinical features of sepsis can be induced by potent pro-in ammatory cytokines, the term systemic in ammatory response syndrome (SIRS) has also been used when describing neonatal sepsis (2). the presence of sequential organ dysfunction (objectively determined by the sequential organ failure assessment (SOFA score) predicts mortality (e.g., sofa score > 4 predicts mortality) and admission to the intensive care unit with death as a nal outcome measure. The presence of organ dysfunction indicates a more complex pathobiology than simply infection with an accompanying in ammatory response (3). Sepsis is classi ed according to the infant's age at the onset of symptoms to: Early-onset sepsis de ned as the onset of symptoms before 7 days of age, although some experts limit the de nition to infections occurring within the rst 72 hours of life (Wynn et al., 2014)(2) and Late-onset sepsis that de ned as the onset of symptoms at ≥ 7 days of age similar to early-onset sepsis, there is variability in the de nition, ranging from an onset at > 72 hours of life to ≥ 7 days of age (Edwards MS, 2004)(4).
Early-onset neonatal sepsis occurs in utero from either transplacental or, more commonly, ascending bacteria entering the uterus from the vaginal environment following membrane rupture.. (5). Premature rupture of membranes (PROM) is a common event in obstetrics that has a major impact in pregnancy outcome. (6).
The overall incidence of neonatal sepsis ranges from one to ve cases per 1000 live births.Estimated incidence rates vary based on the case de nition and the population studied. Globally, neonatal sepsis and other severe infections were responsible for an estimated 430,000 neonatal deaths in 2013, accounting for approximately 15 percent of all neonatal deaths (7).Rates of neonatal sepsis increase with decreasing gestational age (8).The estimated incidence of sepsis (both early-and late-onset) in term neonates is one to two cases per 1000 live births (9).
Group B Streptococcus (GBS) and Escherichia coli are the most common causes of both early-and lateonset sepsis, accounting for approximately two-thirds of early-onset infection (10).Other bacterial agents associated with neonatal sepsis include: Listeria monocytogenes accounts for rare sporadic cases of neonatal sepsis, usually acquired transplacentally (11)).Staphylococcus aureus, including communityacquired methicillin-resistant S. aureus, is a potential pathogen in neonatal sepsis (12). Bacteremic staphylococcal infections in term infants usually occur in association with skin, bone, or joint sites of involvement.Enterococcus is a commonly encountered pathogen among preterm infants. The most common viral causes of sepsis are:Herpes simplex virus associated with substantial morbidity and mortality. Manifestations of viral infections can result in presentations similar to sepsis and might be localised to the skin, eyes, and mouth, involve the CNS, with onset between days 5-9 of life (13). Enterovirus infections might develop meningoencephalitis, myocarditis, and hepatitis, following poor feeding, lethargy, fever, irritability, hypoperfusion, and jaundice (14).
Preterm low birthweight infants have a 3-10 times higher incidence of infection than full-term normal birthweight infants. Immune dysfunction and an absence of transplacentally acquired maternal IgG antibodies in premature infants might increase risk of infection. (15). Clinical manifestations range from subtle symptoms to profound septic shock, (16).
Hematological indices such as total white blood cell count, absolute neutrophil count (ANC), absolute band count (ABC), immature to total white blood cell ratio (I:T ratio) and platelet count are commonly used in the evaluation of neonatal sepsis (18). More recently, advanced white blood cell indices such as mean neutrophil volume (MNV), mean monocyte volume (MMV), and distribution width (NDW; MDW) are emerging as possible additional markers of NS (19). WBC count of < 5000 to 7500/mm3 have been used for the diagnosis of neonatal sepsis. Leucopenia has shown to have low sensitivity (29%) but high speci city (91%) for the diagnosis of neonatal sepsis, Neutropenia has shown to be more predictive of neonatal sepsis than neutrophilia (20). Biomarkers for diagnosis of neonatal sepsis have been discovered that help in the early diagnosis of neonatal sepsis, before the onset of clinical manifestation so that early treatment of sepsis can be started and neonate can be properly managed (21).

Materials And Methods
The present study is a prospective cross-sectional study, was conducted on 40 neonates delivered in Obstetrics and Gynecology department, Minia University Children and Maternal hospital. A total of 80 neonates were included in the study, 40 of them had been admitted in our neonatal intensive care unit have been taken as a study group, in addition to 40 healthy control newborns were selected during follow up in our NICU, during the period from July 2018 to January 2019.
Cases of neonatal sepsis were selected for this study on the basis of standard risk factors, symptoms and signs of neonatal sepsis according to neonatal-speci c SOFA . (3) Neonates born by spontaneous vaginal delivery or cesarean section between gestational weeks 37 and 42, according to ultrasonographic investigations and the new Ballard Score, who were diag-nosed with suspected or proven sepsis, were included in the study. Delivery room data of all mothers and neonates (sex, BW, birth height [BH], birth head circumference [BHC], weeks of gestation [WG], Apgar scores at 1 and 5 minutes after birth, and mode of delivery) were recorded.
Exclusion criteria comprised multiple pregnancies, prematurity (< 37 completed gestational weeks), postmaturity (> 42 completed gestational weeks), small for gestational age (SGA) or large for gestational age (LGA) neonates by week of pregnancy, preeclampsia, gestational diabetes mellitus (GDM), chorioamnionitis, the mother used tobacco during pregnancy, congenital major anomalies, cyanotic congenital heart disease, and negative values of together with C-reactive protein (CRP), and procalcitonin were excluded from the study.
We hypothesized that NLR and PLR ratios in early-onset neonatal sepsis could be used as adjunct diagnostic methods. We calculated that a sample size of 40 in the study group and Another 40 neonates of matchable age and sex, apparently healthy were enrolled in the study as a control group. would allow us to detect differences between the 2 groups. Total blood count was that include hemoglobin, RBCS, red cell indices ( MCV, MCH and MCHC), platelet count and white blood cell count ( total and differential ). It was determined by automated hematology analyzer, CELLTAC G (NIHON KOHDEN CORPORATION). Differential leucocytic count was con rmed by microscopic examination of Lishman stained blood lm and I/T was calculated by the immature /total neutrophil ratio. The NLR was calculated by the neutrophil /lymphocyte ratio, and PLR by the platelets / lymphocyte ratio. ESR was determined by Westergren Method. CRP levels were measured by immunoturbidimetric method, and procalcitonin levels were measured by Electrochemiluminescence immunoassay "ECLIA" method (Roche cobas 6000; Roche Diagnostics GmbH, Mannheim, Germany).
The study protocol was approved by ethical committee, Faculty of Medicine, Minia University. A written consent was obtained from one of parents to agree to participate in the study.
Statistical Analysis All statistical tests done by SPSS version 20 in the form of Descriptive data and Analytical statistics, Descriptive statistics were calculated for the data in the form of Mean and standard deviation for quantitative data. Mean ± SD and Frequency and distribution for qualitative data. In the statistical comparison between the different groups.

Results
This study was conducted with 60 term, appropriate for gestational age (AGA), singleton neonates, of them, 30 were diagnosed with neonatal EOS, and 30 were healthy controls. Comparison between studied patients with EOS and controls regarding the demographic characteristics in (  (Table 4).
We found that Cases had signi cant higher leucocyte count compared to controls (p = 0.004). also Cases had signi cant lower platelet count and lymphocytes compared to controls, (p = 0.04). Also cases had signi cant increase in immature neutrophil count and I/T ratio compared to controls, (p = 0.0001).
Comparing NLR and PLR between cases and controls, cases had signi cant higher values of these ratios rather than controls indicating their valuable role in detection of early-onset neonatal sepsis (p = 0.0001).
Regarding CRP and Procalcitonin, cases had signi cant higher levels of CRP and Procalcitonin rather than controls (p = 0.0001).

Discussion
Neonatal sepsis is a signi cant global health problem associated with high mortality and poor long-term outcomes for survivors particularly in under-resourced settings (22).
In one Egyptian study, the total mortality rate for the proven neonatal sepsis was 51% and 42.9% for EOS and LOS, respectively. Coagulase negative staphylococci were predominant isolates in both EOS and LOS followed by Klebsiella pneumoniae. Also they noticed that most of the bacterial isolates had low sensitivity to the commonly used empiric antibiotics. However, 70.1% exhibited multidrug resistance. Best sensitivities among Gram-positive isolates were found against imipenem, cipro oxacin, vancomycin, and amikacin. (23) We aimed in our study to assess the Platelet to Lymphocyte ratio (PLR) and the Neutrophil to Lymphocyte ratio (NLR) and determine their value as diagnostic markers for detection of EOS in Full-term newborns.
During sepsis or tissue infection Neutrophils are activated, causing their numbers in circulation to rapidly rise (24). Neutrophils are the most abundant leukocyte circulating in the bloodstream, comprising well over 50% of leukocytes, and these cells are particularly adept at phagocytosing and killing microbes (25).
Circulating platelet-neutrophil complexes occur in a diverse range of in ammatory disorders and sepsis). Activated platelets bind to neutrophils in the blood and mediate neutrophil recruitment to sites of injury and infection (26).
Though White blood cell count (WBC) is one of the routinely done diagnostic tests for sepsis work up, was believed to be reliable indicators of infection but now are known to be insensitive and nonspeci c.
Furthermore, a single leukocyte count obtained shortly after birth is not adequately sensitive for diagnosing neonatal sepsis (27).
We found that Cases had signi cant higher leucocyte count compared to controls indicating role of leukocytosis in diagnosis of neonatal sepsis (p = 0.004). also Cases had signi cant lower platelet count and lymphocytes compared to controls, this observation correlates relation of thrombocytopenia and neonatal morbidity as major conscequences of neonatal sepsis (p = 0.04). Also cases had signi cant increase in immature neutrophil count and I/T ratio compared to controls, this indicates the importance of CBC with differential in detecting early-onset neonatal sepsis (p = 0.0001) IN Our study ,Comparing NLR and PLR between cases and controls, cases had signi cant higher values of these ratios rather than controls indicating their valuable role in detection of early-onset neonatal sepsis (p = 0.0001).
In our study CRP is important biomarker in the diagnosis of neonatal sepsis ,that agreed with (Sorsa and  (19) who reported that though, white blood cell (WBC) count ,immature/total leukocyte ratio (IT ratio), absolute leukocyte count and acute phase reactants such as C-reactive protein (CRP), procalcitonin (PC) and interleukin-6 (IL-6) are the most frequently used parameters for the diagnosis of newborn sepsis, these in ammatory markers, however, may be affected by maternal and fetal non-infectious conditions, and their different half-lives may decrease their ability to provide a de nitive diagnosis of sepsis.
Our results show higher incidence of klebseilla in blood culture and these results agreed with (Vergnano et al., 2005) (38) found the same result that laboratory processing of blood cultures has remained static over the past 30 years, despite increasing antibiotic resistance and advances in analyser design.
On the other hand; even though blood culture is said to be the gold standard diagnostic test for sepsis, there are a number of limitations including; unavailability in the majority of developing country, associated technical problem and it takes more than three days to see at least the rst preliminary result .As a result, the diagnosis of neonatal sepsis is based on clinical assessment and the management also rely on empirical treatment protocol which usually results in unnecessary hospital stay, increase irrational use of antibiotics and incur an unnecessary cost for the family (27).
From the results of our study we concluded that:   (WBCS = white blood cells, I/T = immature to total neutrophilic count, NLR = neutrophil to lymphocyte ratio, PLR = platelet to lymphocyte ratio)  (NLR: neutrophil to lymphocyte ratio, PLR: platelet to lymphocyte ratio, PPV: positive predictive value, NPV: negative predictive value, LR+: positive likelihood ratio, LR-: negative likelihood ratio.)