early - onset neonatal sepsis ( eons ) , defined as occurring in the first 3 days of life , continues to be a leading cause of morbidity and mortality in neonates . diagnosis of sepsis is a formidable challenge for neonatal intensive care units ( nicu ) because clinical signs of sepsis are nonspecific , subtle and are observed in other non - infectious conditions . furthermore , bacterial cultures are time - consuming , frequently false negative , especially in neonates , due to low or intermittent bacteraemia , small blood inoculation volumes or intrapartum antibiotics use . recently performed meta - analysis of molecular assays used for early detection of bacteraemia in neonates have yielded disappointing results compared with blood cultures . thus , despite its limitations , blood cultures still remain a  gold standard. to assist in the diagnosis of neonates with suspected sepsis , many laboratory tests including whole blood cell count , differential , immature - to - total neutrophils ( i / t ) ratio , along with acute - phase reactants such as c - reactive protein ( crp ) and procalcitonin ( pct ) , are routinely used . however , so far none of the single laboratory tests can provide reliable and early identification of neonates with sepsis . this lead the recent interest has focused on chemokines , cytokines and acute phase reactants ( and other substances associated with the inflammatory response ) , whose serum levels may reflect the host s immunological status and response to infection [ 46 ] . interleukin 17a ( il-17a ) is a cytokine secreted mainly by a recently characterized distinct subset of helper t cells the th17 cells are considered to be important for immunity to microorganisms that are not eliminated by th1 or th2-cell mediated response . interleukin 17a mediates pro - inflammatory response by triggering the production of many other cytokines , such as il-1 , il-6 and tumor necrosis factor ( tnf ) and orchestrates the response of both innate and adaptive immune cells . neutralization of il-17a exerted a protective effect on sepsis mortality , characterized by decreased bacteraemia and a decreased level of inflammatory cytokines . studies conducted recently in an adult population revealed a significantly higher il-17 production in patients with severe sepsis than in healthy controls [ 10 , 11 ] . c5a is considered a central mediator in the immunopathogenesis of sepsis , as it modulates other systems , including the coagulation cascade and the release of proinflammatory cytokines . excessive c5a production , in turn , elicits numerous harmful effects [ 7 , 15 ] . on the other hand , it has been demonstrated that blockage of c5a greatly improve the survival of rodents during sepsis . significantly enhanced levels of c5a have been found in septic shock in adult patients compared with healthy individuals , but none of the study has evaluated this anaphylatoxin in the setting of neonatal sepsis , so far . regulated on activation , normal t cell expressed and secreted ( rantes ) , also known as chemokine ( c - c motif ) ligand 5 ( ccl5 ) , mediates the trafficking and homing of t helper cells , monocytes and eosinophils into inflammatory sites . an inverse correlation has been reported between the levels of rantes and disease severity , and mortality in patients with sepsis and lower levels of this chemokine were found in non - surviving patients , suggesting its beneficial pro - inflammatory role in host response to infection . a few studies reported rantes levels in infected neonates [ 1922 ] including those with eons [ 20 , 22 ] . most of them documented lower rantes levels in infected neonates compared to non - infected controls . the aim of this preliminary study was to investigate serum levels of il-17a , anaphylatoxin c5a and chemokine rantes in neonates with eons and those non - infected , and to evaluate the potential utility of these novel markers for early diagnosis of neonatal sepsis . a total of 68 neonates were enrolled prospectively in the neonatal intensive care unit ( nicu ) at the nicolaus copernicus hospital in olsztyn and in the tertiary nicu at the medical university of warsaw . neonates eligible for inclusion in the case group had to have clinical signs of early - onset neonatal sepsis ( eons ) developed within 72 hours of life and/or history of factors associated with increased risk for infection . clinical signs of sepsis were defined as the presence of three or more of the following clinical manifestations , without any other obvious pathological cause : temperature instability ( core temperature  38.5 or  36c ) ; respiratory symptoms ( apnoea , tachypnoea with respiratory rate > 60 per minute , cyanosis , need for high ventilator settings or oxygen ) ; cardiovascular symptoms including hypotension ( blood pressure < 5 percentile for age ) , tachycardia ( heart rate > 160 per minute ) , bradycardia ( heart rate < 80 per minute ) , or poor perfusion ; neurological symptoms ( hypotonia , hyporeflexia , irritability , lethargy and seizures ) ; gastrointestinal symptoms ( poor feeding , abdominal distension , green or bloody residuals , vomiting ) . risk factors were : maternal chorioamnionitis , mothers with inadequately treated group b streptococcus ( gbs ) colonisation , mothers non - tested for gbs with premature rupture of membranes ( prom ) for > 18 hours , maternal fever during labour or premature labour . newborns with the above - mentioned clinical findings and/or risk factors have had sepsis screening laboratory tests , including : acute phase reactant c - reactive protein ( crp concentrations > 5 mg / l were considered abnormal ) , white blood cell ( wbc ) differential count ( wbc > 35  10/l or < 5  10/l was considered abnormal ) and immature to total neutrophil ratio ( i / t ratio > 0.2 was considered abnormal ) , and platelet counts ( plt < 80  10/l was considered abnormal ) . wbc , neutrophil and immature wbc counts were measured using an sysmex xt-1800i automated hematology analyzer ( sysmex america , mundelein , usa ) . the plasma levels of crp were determined using roche cobas integra 800 full - automated analyzer ( roche diagnostics , mannheim , germany ) . at the same time , an additional blood sample was provided for quantitative analysis of il-17a , c5a and rantes . inclusion criteria for the case group were the presence of clinical signs of sepsis and/or the presence of risk factors plus at least two abnormal laboratory test results . diagnosis of respiratory distress syndrome , necrotizing enterocolitis , meconium aspiration syndrome , hypoxia , intracranial haemorrhage or congenital heart disease was ruled - out in all neonates included in the study . the variables of each case recorded included newborn s demographics , prenatal and postnatal history , physical examination findings , vital signs , routine blood test , microbiologic culture results and applied antibiotic therapy . the criteria for inclusion to the control group were neither evidence of infection in neonate nor perinatal risk factors and a whole blood sample collected within the first 72 hours of life available for assessment of il-17a , c5a and rantes . data recorded for healthy neonates included : gestational age , gender , delivery mode , birth weight , five - minute apgar score and crp level . the study was conducted in accordance with the declaration of helsinki and written informed consent was obtained from the parents or legal guardians . the study protocol was approved by the ethical committee of the children s memorial health institute . for the il-17a , c5a and rantes analysis 1 ml of the venous blood was collected into test tubes without anticoagulants and allowed to clot for 30 minutes and then centrifuged at 4c for 10 minutes at 1000  g. each neonate s serum sample was separated into 3 aliquots and stored immediately at 20c until analysis . interleukin 17a , c5a and rantes were measured using commercially available solid - phase sandwich enzyme - linked immunosorbent assay ( elisa ) kits ( human il-17a elisa development hrp kit , mabtech ab , nacka strand , sweden for il-17a ; bd opteia human c5a elisa kit ii , becton , dickinson and company bd biosciences , san jose , ca , usa for c5a ; human rantes elisa construction kit , antigenix america inc . , melville , ny , usa for rantes ) . the detection limit was 4 pg / ml , 0.047 ng / ml and 0.016 ng / ml for il-17a , c5a and rantes , respectively . all clinical specimens were transported frozen on dry ice and tested simultaneously to ensure the uniformity of measurements . assays for il-17a , c5a and rantes were performed in the department of clinical microbiology and immunology , the children s memorial health institute in warsaw . categorical variables were expressed as number and percentage . normally distributed continuous variables were summarized as mean  standard deviation ( sd ) , whereas other continuous variables were presented as median and 25 to 75 percentiles ( interquartile range , iqr ) . statistical significances of the differences in serum c5a and rantes levels between different study groups were assessed by the mann - whitney u test . for correlation analysis , spearman a total of 68 neonates were prospectively enrolled in this preliminary study within the first 72 hours after birth . eighteen neonates with clinical signs and symptoms of systemic infection formed the eons group , whereas 50 neonates without evidence of infection were classified into the control group . as compared with non - infected neonates , newborns with eons were more likely to have a lower gestational age . the groups differed also in delivery mode and , as expected , in crp concentration ( table 1 ) . there was no difference according to birth weight , length or male / female ratio . demographic and clinical characteristics of septic neonates ( eons group , n = 18 ) and non - infected neonates ( control group , n = 50 ) me  median ; irq  interquartile range ; eons  early - onset neonatal sepsis ; crp  c - reactive protein p - values by student t test , mann - whitney u test or fisher s exact test as appropriate ; bold indicated statistical differences . measured between 23 and 25 hour of life . regarding neonates in the eons group , the diagnosis was based on clinical grounds supported by laboratory tests . no patients had a positive blood culture . at the time of the sepsis episode 9 neonates presented respiratory distress and 6 of them had a diagnosis of pneumonia . detailed characteristics of neonates with eons i / t ratio  immature to total neutrophil count ; me  median ; iqr  interquartile range ; gbs  group b streptococcus ; prom  premature rupture of membranes . diagnosis of pneumonia in all cases . defined as maternal fever ( > 38oc ) , and at least two of the following criteria : maternal leukocytosis ( wbc > 15  109/l ) , maternal tachycardia ( heart rate > 100 per minute ) , foetal tachycardia ( heart rate > 160 per minute ) , uterine tenderness and/or foul odour of the amniotic fluid . at the time of sepsis investigation serum levels of c5a in the eons group were significantly higher compared to those in the non - infected group ( median 65.35 vs. 50.4 ng / ml , p = 0.034 ; fig . 1a ) , whereas no significant difference in rantes levels between the two groups were found ( median 188.95 vs. 195.2 ng / ml ; p = 0.444 ; fig . scatter plots representing c5a ( a ) and rantes ( b ) levels in serum of neonates . the c5a levels measured in septic neonates ( eons ) vs. non - infected neonates ( healthy controls ) showed a significant difference ( p = 0.034 ; by mann - whitney u test ) levels of il-17a were under the detection limit ( < 4 pg / ml ) in both eons and non - infected neonates . il-17a was excluded from further analysis because of having values below the assay working range , which would not enable an examination as a continuous variable . a separate analysis was performed to determine whether there was a difference between premature and term neonates as well as among neonates according to delivery mode . concentrations of c5a and rantes did not differ between premature and term neonates ( median c5a level 46.1 vs. 52.7 ng / ml ; p = 0.397 and median rantes level 254.1 vs. 185.7 ng / ml ; p = 0.367 , for premature and term neonates , respectively ) . similarly , no significant difference was observed according to delivery mode ( median c5a level 51.4 vs. 56.2 ng / ml ; p = 0.748 and median rantes level 207.6 vs. 171.6 ng / ml ; p = 0.402 , for vaginal and non - vaginal delivery , respectively ) . correlation analysis revealed a significant positive correlation between serum levels of c5a and crp ( spearman r = 0.33 ; p = 0.010 ) . the early identification of neonates with eons is critical , because it allows clinicians to implement suitable and more effective medical intervention improving the outcome . reliable ruling - out of sepsis is also important to avoid exposure to broad - spectrum antibiotics ( which , even if for only a few days , might foster antibiotics resistance patterns ) and the long - term effects of antibiotic overuse in patients . the lack of a reliable test for early diagnosis of eons forces a search for new biomarkers . in this preliminary study , 3 novel markers such as c5a , il-17a and rantes were measured in neonates with eons and non - infected newborns for their potential use in the diagnosis of early - onset neonatal sepsis . in the current preliminary study , for the first time significantly higher levels of c5a were found in neonates with eons compared with non - infected cases . these results are in accordance with the recent study by unnewehr et al . who showed a significant increase of c5a concentrations in adult patients during septic shock . taken together , these results and data from experimental models linking elevated c5a with immunoparalysis , multi - organ failure , imbalances in the coagulation system   and the apoptosis of thymocytes , suggest that c5a increases in early phases of infection and may be an important factor for the inflammatory process in the pathogenesis of sepsis . regarding il-17a , significantly increased production of this interleukin was observed in two previous studies conducted in adult septic patients [ 10 , 11 ] , while little is known about il-17a levels in neonates , especially those with eons . contrary to the findings in adults , in the current study il-17a concentrations were under the detected level in both eons and non - infected cases . nevertheless , the results are in accordance with the previous study by papoff et al . comparing il-17 expression in body fluids from 13 sick and 7 healthy neonates admitted to nicu . undetectable levels of il-17 were found in all plasma samples of infected neonates as well as those with inflammatory non - infectious diseases and controls . this is in line with a recent study by caron et al . , who examined neonatal production of il-17 in mixed mononuclear cells ( mmcs ) isolated from umbilical cord blood and found severely depressed il-17 production by neonatal mmcs compared to adults . in addition , reduced intracellular formation of il-17 , and diminished il-17 response to both group b streptococci ( gbs ) and escherichia coli , were observed . taking into account the fact that in healthy children serum levels of il-17 increase with age , low or undetectable il-17 levels observed in neonates may mirror the immaturity of the neonatal immune system to launch an effective th17 response . most of the previous studies showed lower rantes concentration in septic neonates compared with healthy subjects [ 1922 ] , though diametrically opposite results have also been reported in the setting of eons . in the current study the median concentration of this chemokine was lower in eons group then in non - infected neonates , but the difference did not reach statistical significance . this discrepancy in the results between the current and previous studies may be attributed to the relatively small sample size , differences in timing of evaluation ( early vs. late onset - sepsis ) or varied patient population ( including term vs. premature neonates ) . it must be also underlined , that at the time of sepsis onset , none of the septic neonates enrolled into the current study had significant thrombocytopenia and because platelets are principal reservoir of rantes in peripheral circulation , this may account for the lack of significant difference in chemokine concentration between the groups . confirmation of this fact are data obtained in the esgni-001 study in which the percentage of positive microbiological blood culture among patients with symptoms of bloodstream infections was 15% in european countries . due to ethical reasons the marker evaluations were performed in a single serum sample collected within the first 72 hours of life . serial measurements of the parameters could be more informative , especially when considering their use for the prognosis or treatment monitoring . in conclusion , the results of our study obtained in this small number of patients are very preliminary nonetheless showed that c5a is a very interesting biomarker . thus , there is a strong need for further studies evaluating the dynamics of c5a and its sensitivity and specificity in the diagnosis of eons on a large number of neonates . moreover , it is also necessary to differentiate the usefulness of c5a determination between eons and other pathological conditions , such as necrotizing enterocolitis , meconium aspiration syndrome , hypoxia and respiratory failure of varying etiology . we conclude that such studies will help to assess whether this anaphylatoxin could be useful laboratory tool for differential diagnosis of infectious vs. non - infectious systemic inflammatory response .
the aim of the present study was to investigate serum levels of novel markers : interleukin 17a ( il-17a ) , anaphylatoxin c5a and chemokine regulated upon activation normal t - cell expressed and secreted ( rantes ) in neonates with clinically suspected early - onset neonatal sepsis ( eons ) , and to compare their values with those of non - infected neonates . 
 eighteen neonates with clinical signs and symptoms of eons were enrolled in this study . 
 fifty healthy , non - infected neonates served as the control group . in all neonates 
 serum levels of il-17a , c5a and rantes were measured by solid - phase sandwich enzyme - linked immunosorbent assay ( elisa ) . at the time of investigation serum levels of anaphylatoxin c5a 
 were significantly higher in neonates with clinical symptoms of eons than in non - infected neonates ( median 65.35 vs. 50.4 ng / ml , p = 0.034 ) , whereas levels of rantes were similar and levels of il-17a were under detection limit of the method . based on these preliminary results , serum levels of c5a 
 may be a useful marker of inflammation in early onset neonatal sepsis . because traditional methods of microbiological diagnostics in eons are frequently unsuccessful 
 , the search for an alternative laboratory biomarkers is of great clinical importance . 
 thus , there is a strong need for further studies evaluating usefulness of this anaphylatoxin in eons diagnosis on a larger group of patients .