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Cite this article: Ronchetti MP, Bersani I, Piersigilli F, Auriti C (2017) Neonatal Sepsis Arch Paediatr Dev Pathol 1(3): 1015.

*Corresponding authorCinzia Auriti, Department of Neonatology, Neonatal Intensive Care Unit, Bambino Gesu’ Children’s Hospital, Piazza S. Onofrio 400165 Rome, Italy, Tel: 390668592427; 393357050147; Email: cinzia.

Submitted: 10 September 2017

Accepted: 25 September 2017

Published: 27 September 2017

Copyright© 2017 Auriti et al.

OPEN ACCESS

Keywords•Neonatal sepsis•Preterm neonates; Mortality

Review Article

Neonatal SepsisMaria Paola Ronchetti, Iliana Bersani, Fiammetta Piersigilli, and Cinzia Auriti*Department of Neonatology, Bambino Gesu’ Children’s Hospital (IRCCS), Italy

Abstract

Despite the recent advances in perinatal care, neonatal sepsis still represents a challenging clinical condition increasing the risk for neonatal morbidity and mortality, especially in preterm neonates. The aim of the present paper was to provide an overview about the most recent data about the pathogenetic, biochemical, laboratory, microbiologic, and clinical features of neonatal sepsis.

ABBREVIATIONSBW: Birth Weight; CSF: Colony Stimulating Factor; ELBW:

Extremely Low Birth Weight; EOS: Early Onset Sepsis; GA: Gestational Age; GAS: Group A Streptococcus; GBS: Group B Streptococcus; IL: Interleukin; IFN gamma: Interferon gamma; LBW: Low Birth Weight; LOS: Late Onset Sepsis; MIF: Macrophage Inhibiting Factor; MBL: Mannose Binding Lectin; MRSA: Methicillin-resistant Staphylococcus aureus; NO: Nitric Oxide; PAMPs: Pathogens Associated Molecular Patterns; PRRs: Pattern Recognition Receptors; TLRs: Toll-like Receptors; TNF alfa: Tumor Necrosis Factor alfa; VLBW: Very Low Birth Weight

INTRODUCTION Neonatal sepsis is a systemic condition, with bacterial,

viral, or fungal etiology, potentially associated with serious complications and high rates of mortality.The incidence of neonatal sepsis varies between different hospitals and geographic areas. In industrialized countries, the incidence of sepsis in at term neonates with appropriate weight is around 0.1%, but it increases up to 10% in infants with birth weight (BW) between 1000 and 1500 g, to 35% in those with BW <1000 g and up to 50% in those with BW <750 g (1-3). The higher incidence among very low birth weight (VLBW) infants (BW <1500 g) reflects the major role of an immature immune system in conditioning the risk of neonatal sepsis.

PATHOGENESIS OF NEONATAL SEPSISThe observed variability in the reported incidence of

infections in neonates still remains unclear. Biological and or genetic factors could intervene in neonatal predisposition to infections. The neonatal reduced immune defence, genetic factors regulating the expression of immunity factors, the complex interactions between the infecting microorganism and the host response and highly invasive procedures related to the health care, all together could modulate the neonatal susceptibility to infections and partially explain this variability.

Sepsis is a clinical condition, initiated by a pathogen, which leads to disturbed immune, inflammatory, and coagulation

homeostasis (Figure 1) [1-3]. The evolution of the disease and clinical symptoms are dependent upon a complex and delicate balance between the pro inflammatory and anti-inflammatory factors. The inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-15, IL-18, Macrophage Inhibiting Factor-MIF), and growth factors (IL-3, CSFs) and their secondary mediators, including Nitric Oxide (NO), tromboxanes, leukotrienes, platelet activating factor, prostaglandins and complement, cause activation of the coagulation cascade, the complement cascade, and the production of prostaglandins, leukotrienes, proteases, and oxidant. Most of the short and long term complications (from septic shock to respiratory and neurological sequelae of neonatal sepsis) are strictly associated to the effects of these mediators, not counterbalanced by an adequate synthesis of anti-inflammatory cytokines as TNFsr,IL-1ra,IL-1rII, IL-10,TGF-β2 [4,5].

Neonates, especially preterm infants, are relatively immune compromised, with unbalanced immune response to micro-organisms invasion due to immaturity of the innate and adaptive

Figure 1 It shows the increase of cytokines levels occuring much earlier than the one of conventional hematological and biochemical markers of infections.


Auriti et al. (2017)Email:

Arch Paediatr Dev Pathol 1(3): 1015 (2017) 2/15

immune systems. Moreover, the significant placental passage of maternal antibodies begins around 20 weeks of gestation. As a result, preterm neonates have lower IgG levels than term neonates with an increased risk for infection development, especially LOS.

The innate immune response is the most important first line defence mechanism in newborn infants, producing an immediate immunological response without previous exposure, to a specific pathogen. It works through the recognition of the pathogens associated molecular patterns (PAMPs- bacterial cell wall components, flagellin, nucleic acids) by specific pattern recognition receptors (PRRs), which are present on the cell surface, within intracellular vesicles, and in the cytoplasm of multiple cell types. Three major families of receptors have been described: soluble receptors (Mannose Binding Lectin, MBL), scavenging receptors (expressed by myeloid cells, macrophages and dendritic cells and certain endothelial cells), and signalling receptors (TOLL like receptors-TLRs). They identify and respond to PAMPs with the production of cytokines and pro inflammatory responses that activate the adaptive immune system. Neonatal cells have a decreased ability to produce inflammatory cytokines, especially Tumor Necrosis Factor and Interleukin 6 when compared to adults. In addition, they induce IL10 production, which inhibits the synthesis of pro inflammatory cytokines [6-8]. Cellular immune functions are also reduced: neutrophils show a decreased expression of adhesion molecules as well as a decreased response to chemotactic factors, and dendritic cells produce low levels of IL12 and Interferon gamma (IFN). The reduction in cytokines production results in decreased activation of natural killer cells [9-13].

One of the soluble receptors, the Mannose Binding Lectin (MBL), is a crucial molecule of the innate immune system recognition receptors. MBL recognizes and binds to sugar moieties on the surface of bacteria, viruses, fungi, and parasites. Its binding causes these microorganisms to agglutinate and allows phagocytic clearance of pathogens as well as lectin-complement pathway activation, through MBL-associated proteases. As part of the innate immune system, MBL is considered particularly important in the vulnerable period of infancy, before the adaptive immune response provides adequate specific immune protection [14-16]. In neonates, MBL levels are low at birth and increase in the first weeks or months of life. MBL levels are also lower in preterm than in term infants and are related to gestational age [17]. MBL shows a remarkable genetic variability, due to polymorphisms of both the promoter and the exon domains of its gene: some haplotypes, about 10% in total, are associated with MBL insufficiency, defined as a serum level below 0.5-0.7 μg per ml.

We have reported that low MBL serum levels on admission to the NICU increase the risk of neonatal sepsis in critically ill neonates independently from gestational age and the increased risk is independent of invasive procedures Other Authors have observed a similar association. Moreover, in 365 critically ill neonates (261 without infection) we have found a significant correlation between the initial serum MBL level measured on admission and the peak level reached during the infection: low levels on admission were associated with low peak levels during

infection, without an increase in mortality [18-20]. Our findings support the hypothesis that genetic and/or developmental variations of some components of the innate immune system are likely to play an important role in the defence mechanisms from severe infections in high risk newborn infants [19-21]. In neonatal clinical practice it is difficult to define a cut-off value that identifies MBL deficiency and a subsequent risk of infection. Measuring MBL levels alone appears not sufficiently sensitive and specific in defining the risk of LOS. Given our finding that MBL contributes notably to the risk of sepsis, independently from invasive procedures and GA, we suggest that MBL levels could be included in an algorithm designed to define the risk of sepsis, so that preventive measures and unconventional therapies could be adjusted according to the individual neonate’s needs. Notwithstanding a cautionary note regarding the possible role of MBL in ischemia/perfusion pathologies (in example Necrotizing Enterocolitis [22], the recently available recombinant MBL may offer a new approach for preventing infection in premature neonates. Further studies should evaluate how MBL levels and the MBL2 genotype influence the potential long-term sequelae of prematurity.

The adaptive branch of the immune system is designed to eliminate specific pathogens. In newborns, the adaptive immune system slowly increases its functions toward an adult-like response, minimizing the otherwise overwhelming inflammatory response that occurs when infants transition from a sterile to a colonized environment. Decreased cytotoxic function (strong T-helper 2 polarization with decreased IFN-gamma production), lack of isotype switching, and overall immaturity and decreased memory (because of limited pathogen exposure at time of birth), reduce the neonate’s ability to respond effectively to infections. The reduction of cell mediated immunity increases the risk of infections from intracellular pathogen, such as Listeria, Salmonella, Herpes viruses, Cytomegalovirus and Enterovirus.

Neonates have passively acquired antibodies via placental transfer. A significant increase of transplacental passage of maternal antibodies begins around 20 weeks of gestation and serum levels of immunoglobulins G (IgG), particularly IgG1 and IgG2 subclasses, are inversely related to GA, limiting the functional ability to respond to certain pathogens. The neonate receives no immunoglobulins of the IgA and IgM classes in fetal life. Specific bactericidal and opsonic antibodies against gram negative bacteria are predominantly in the IgM class. Thus, the newborn infant lacks optimal humoral protection from infection with enteric organisms. Minimal IgG is transported to the fetus in the first trimester, whereas fetal IgG rise in the second trimester from approximately 10% at 17 to 22 weeks’ gestation to 50% at 28 to 32 weeks’ GA [23,24]. Consequently, preterm infants have not an efficient humoral protection against a number of pathogens, whereas term infants will often be protected against most vaccine-preventable neonatal infections through transplacental passage from the mother’s serum. Histologic studies have also demonstrated that the marginal zone of the spleen is not fully developed until 2 years of age, increasing infant’s susceptibility to encapsulated bacterial infections (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis) [25]. Finally, transfer of IgA, IgG, cytokines, and antibacterial peptides present in human milk may be compromised, especially in premature




babies. The lack of secretory IgA decreases the ability of the neonate to respond to environmental pathogens [26].

Non cellular components of the Innate Immune Response include inflammatory response proteins, such as complement, acute phase reactants, cytokines, chemokines, coagulation proteins and vasoactive substances. Actions of complement include opsonization and killing of pathogens, alteration of the vascular tone to facilitate the recruitment and activation of leukocytes, initiation of the coagulation cascade, and pro inflammatory cytokine production. Neonates also exhibit decreased levels of complement-mediated opsonic capabilities when compared to adults. There is no transfer of complement from the maternal circulation. The foetus synthesizes complement as early as the first trimester. Full term infants have slightly diminished classic pathway complement activity and moderately diminished alternative pathway activity [27,28]. Complement levels increase with increasing GA, but are only about 50% of adult levels at term. Reduced complement levels are associated with deficient opsonization and impaired bacterial killing. Although both pathways seem to be capable of being activated, there may be variations in their activation level. In addition, profound C9 deficiency has been observed in neonates, reducing the ability to form bacteriolytic C5b-9 (m), which will increase the risk of acquiring severe invasive bacterial infections [28,29].

EARLY-ONSET SEPSISEarly-onset neonatal sepsis (EOS) is variably defined, but the

most widely used definition is maternal-fetal transmitted sepsis, diagnosed within the first 72 hours of life [30,31]. However, some authors define EOS as sepsis developing within 7 days of life. EOS are caused by maternal-fetal transmitted organisms, via trans-placental or ascending vaginal routes. The most common etiologic agents are summarized in Table 1.

The etiologic agents responsible for neonatal sepsis in the United States have changed over time [32,33].

The Group B Streptococcus (GBS) is the most common organism inducing EOS especially among at term neonates, although according to the Centers for Disease Control and Prevention (CDC), rates of early-onset invasive GBS disease have declined by 80%, since the CDC prevention guidelines were first published [34,35]. GBS are gram-positive encapsulated bacteria for which 10 different serotypes have been identified; serotype III strains are responsible for most of diseases (54%) [36]. GBS commonly colonize the gastrointestinal (GI) and genital tracts, with rates up to 20% in the adult population. Transmission occurs late in pregnancy or during labor and delivery, and the likelihood of disease, as well as the severity, has been associated with the density of recto-vaginal carriage [37,38]. GBS has different virulence factors which determine its ability to cause invasive disease: the capsular polysaccharide, which helps evade phagocytosis; pili, which allow adherence of GBS to the host’s epithelial cells as well as transepithelial migration; and the C5a peptidase, which inhibits human C5a, a neutrophil chemo attractant produced during complement activation.

Among infected newborns, clinical manifestations develop very early after delivery and most infants will have signs of

respiratory distress and cardiovascular instability. Infants with early-onset GBS are at increased risk for meningitis. Rapid deterioration of the clinical status is expected unless prompt antibiotic management is started. Risk of death is inversely related to gestational age (GA), with mortality of 20% to 30% among infected infants of less than 33 weeks’ GA, compared with a mortality of 2% to 3% in full-term infants [39].

Other common causative pathogens of EOS are Gram-negative bacteria (25% Escherichia coli) [40,41]. Sepsis caused by E. coli has increased in recent years, mainly affecting preterm newborns weighing less than 2500 g at birth, and is considered the most common cause of EOS in this weight group. E. coli is frequently associated with severe infections and meningitis and it has become the leading cause of sepsis-related mortality among VLBW infants (24.5%) [8]. Together, GBS and E. coli account for about 70% of cases of EOS in the neonatal period.

Escherichia coli, a gram-negative rod that commonly colonizes the maternal urogenital and GI tracts, is considered the second most common cause of neonatal sepsis in term infants and the most common cause in VLBW neonates with rates of 5.09 per 1000 live births [42-44]. The antigenic structure of E. coli is represented by multiple antigens (O), (K), and (H), which in combination account for the genetic diversity of the bacteria. Strains with the K1 antigen have been associated with the development of neonatal sepsis, meningitis, as well as a more aggressive presentation, with a higher risk of thrombocytopenia and death in the first days of life, when compared with K1-negative strains. Several US studies have shown high rates of ampicillin resistance in E. coli strains that infect newborns, when the infection is vertically transmitted. Although some studies have shown an association between intrapartum antibiotic exposure and ampicillin-resistant E. coli, ampicillin resistance

Table 1: Microbial pathogens and risk factors for EOS and LOS.

Neonatal sepsis

Microbial pathogens Risk factors

EOS • Group B streptococci• Escherichia coli• Streptococcus viridans• Enterococci• Staphylococcus aureus• Pseudomonas aeruginosa• Other gram-negative bacilli

• Maternal Group B streptococcal colonization• Chorioamnionitis• Premature rupture of membranes• Prolonged rupture of membranes (> 18 h)• Preterm birth (< 37 weeks)• Multiple gestation

LOS • Coagulase-negative Staphylococci• Staphylococcus aureus• Candida albicans• Escherichia coli• Klebsiella pneumoniae• Enterococci• Pseudomonas aeruginosa• Group B streptococci

• Prematurity• Low birth weight• Prolonged indwelling catheter use• Invasive procedures• Ventilator associated pneumonia• Prolonged antibiotics




has increased throughout the community and a direct link between intrapartum use of ampicillin and the higher likelihood of resistance has not been established [40,44-46].

Although less common, Listeria monocytogenes is associated with invasive disease in the newborn, spontaneous abortions or stillbirth if acquired during pregnancy. Although evidence of disease has been described since the end of the 19th century in several animal species, the first human case of Listeria was reported in 1929 [47], and the first perinatal case in 1936 [48]. Listeria is a facultative anaerobic, gram-positive bacterium found in soil, decaying vegetation, fecal flora, and raw unprocessed food [49-51]. Multiple virulence factors allow Listeria to escape the immune system, including listeriolysin, which helps the organism avoid the oxidative stress of phagolysosomes, allowing intracellular replication. Listeria proteins Act A, phospholipase C, and lecithinase allow polymerization of actin and lysis of phagosomal membranes, enabling cell-to-cell transmission [52,53]. Pregnant women have 17% higher risk of Listeria infection than not pregnant women, and infection has been associated with spontaneous abortions and stillbirths [54]. As EO GBS infections, also EO Listeria infections present with respiratory distress, sepsis, and meningitis. In severe cases, patients may present with a granulomatous rash (small patches with erythematous base), known as granulomatosis infantisepticum. Most cases of neonatal Listeria are caused by serotypes 1, 2, and 4, with the latter serotype responsible for almost all cases of meningitis [54]. Suspicion for Listeria sepsis should be increased in ill infants of mothers who have consumed raw milk, unpasteurized cheeses, or other unprocessed food products contaminated with the organism [50,51].

Other less common but important pathogens associated with EOS include other streptococci (Streptococcus pyogenes, viridans group streptococci, Streptococcus pneumoniae), enterococci, staphylococci, and non typable Haemophilus influenzae. Streptococcuspyogenes (group A Streptococcus [GAS]) was once the predominant organism responsible for neonatal sepsis. Although overall incidence has decreased significantly, severe cases of EO GAS continue to be reported. A recent literature review identified 38 cases of neonatal GAS sepsis (24 with EOS). Patients were most likely to present with pneumonia and empyema (42%) or toxic shock syndrome (17%); 70% of the isolates were M1 serotype and they were all susceptible to penicillin. Mortality was estimated to be 38% among patients with EOS [55]. The presentation of pneumococcus, groups C and G streptococci, and viridans streptococci neonatal sepsis is very similar to GBS infection, and transmission seems to be secondary to bacterial colonization of the maternal genital tract [56-60].

Enterococcal EOS is usually mild compared with LOS and is characterized by either a mild respiratory illness or diarrhea without a focal infection. Enterococcus faecalis is more frequently isolated than Enterococcus faecium, and most of the isolates remain ampicillin susceptible [61]. Although nontypable Haemophilus influenzae frequently colonizes the maternal genital tract, neonatal infection is relatively rare, but with high mortality rates, especially in preterm neonates [62].

Hershckowitz and colleagues reported a cluster of 9 cases with 3 deaths; similar high mortality rates were reported in a series by

Takala and colleagues [63,64]. The overall mortality rate of EOS is 15 to 40% (in EO GBS infection it is 2 to 30%). Documented mortality among very premature infants (<32 weeks’ GA) with EOS is higher, being over 30% of infected babies [65,66].

LATE ONSET SEPSISThe increased survival of preterm low birth weight infants,

particularly those who are VLBW, with need for prolonged hospitalization and use of invasive procedures and devices, especially long-term intravascular catheters, results in ongoing risk of infection. Late-onset sepsis (LOS) usually arises from nosocomially acquired organisms. LOS are diagnosed from 72 hours until the 90th day of life, whereas late-late-onset neonatal sepsis (LLOS) are diagnosed after 90 days of life. Late onset GBS infection is diagnosed beginning from 7 days of life [11]. Rates of LOS are most common in preterm infants with a reported incidence ranging from 15 to 30 cases per 1000 live VLBW births

The incidence of neonatal sepsis varies between different hospitals and geographic areas [67-74]. In the UK, the incidence of LOS is approximately 8/1000 live births and affects approximately 7% of neonatal unit admission [75]. Studies from the NICHD NRN report that approximately 21% of VLBW infants developed one or more episode of blood culture-confirmed LOS, with rates inversely related to gestational age (GA) (58% at 22 weeks’ GA and 20% at 28 weeks’ GA) [12-13]. In a multicentric Italian study an incidence density of 6.9 episodes per 1000 patient-days has been recorded [76]. Intrapartum antibiotic prophylaxis seems not to affect the rate of LOS. VLBW preterm infants as well as babies with birth defects needing surgery are at particular risk for LOS, because of prolonged hospitalization and prolonged use of indwelling catheters, endotracheal tubes, surgical interventions, and other invasive procedures. Several studies have documented rates of LOS from 1.87 to 5.42, with decreasing rates as BW increases [77,78]. Coagulase-negative staphylococci (CoNS) have emerged as the most commonly isolated pathogens among VLBW infants with LOS. Meningitis is more frequently a feature of LOS than EOS. Candida infections are relatively frequent in extremely low birth-weight babies (ELBW), but pathogen burden varies between countries, as well as between hospitals in the same country [79].

Mortality of LOS is 10 to 20% (in late-onset GBS infection is about 2-6%) [78]. The case fatality rate is 2 to 4 times higher in low birth weight (LBW) (BW <2500 g at birth) than in full-term infants. The Turkish Neonatal Society Nosocomial Infections Study Group reported that LOS-related mortality was 24.4% of infected babies, with evolution in septic shock in 1.3% of patients and associated mortality peaking to 71 % in extremely low birth weight (ELBW) infants [80,81].

LOS is largely caused by organisms acquired from the environment after birth. CoNS has emerged as the single most commonly isolated pathogen among VLBW infants with LOS and is associated with 22% to 55% of LOS infections among VLBW infants [82,83].

Staphylococcus aureus is associated with 4%to 8% of LOS [84]. Staphylococcus commonly colonizes the human skin and mucous membranes and is capable of adhering to plastic surfaces with the subsequent formation of biofilms. These biofilms protect the




bacteria from antibiotic penetration and can produce substances that will help them evade the immune system. Although CoNS infections are usually secondary to Staphylococcus epidermidis, other strains such as Staphylococcus capitis, Staphylococcus haemolyticus, and Staphylococcus hominis have also been reported [85].

Methicillin-resistant Staphylococcus aureus (MRSA) has been isolated in 28% of staphylococcal infections in preterm neonates with no significant differences between MRSA and methicillin-susceptible organisms in terms of morbidity, mortality, and length of hospital stay. Overall, 25% of infants infected with MRSA die, with no significant difference in death rates between infants infected with MRSA or methicillin-susceptible Staphylococcus aureus [86].

Gram-negative organisms are associated with about one-third of LOS cases, but 40% to 69% of deaths due to sepsis in this age group. Transmission occurs from the hands of health care workers, colonization of the gastrointestinal tract, contamination of parenteral nutrition or formulas, and bladder catheterization devices [87,88]. The most common gram-negative organisms isolated include E. coli, Klebsiella, Pseudomonas, Enterobacter, Citrobacter, and Serratia [30].

In some case series, Klebsiella is recognized as the most common gram-negative agent associated with LOS, ranging from 20% to 31% of cases [89,90]. Infections caused by Pseudomonas have been associated with the highest mortality [91]. Citrobacter is uniquely associated with brain abscesses, but dissemination can occur to other organs. Its ability to survive intracellularly has been linked to the capacity of creating chronic central nervous system infections and abscesses [92,93].

Infections caused by Candida species are the third leading cause of LOS in premature infants. Risk factors of infection include low birth weight, use of broad-spectrum antibiotics, male gender, and lack of enteral feedings [79]. Candida albicans and Candida parapsilosis are the species most commonly associated with disease in neonates [94]. Candida easily grows in blood culture media, but its isolation may require larger volumes of blood than normally obtained in neonates and therefore multiple cultures may be necessary to document infection and clearance. Among those with a positive cerebrospinal fluid culture, as many as 50% will have a negative blood culture; the discordance of blood and cerebrospinal fluid cultures underscores the need for a lumbar puncture (LP). Prompt removal of contaminated catheters is recommended based on the ability of Candida species to create biofilms, as well as better survival rates and neurodevelopmental outcomes in patients who had early removal and clearance of the infection [79].

Poor outcomes, including higher mortality rates and neurodevelopmental impairment, have been associated with the ability of the organisms to express virulence traits, such as adherence factors and cytotoxic substances [95]. HSV is a potentially devastating cause of late-onset neonatal infection. HSV should be included in the differential diagnosis and treatment strategy of newborns who present with signs and symptoms of sepsis, especially after the first few days of life [96].

RISK FACTORS FOR NEONATAL SEPSIS

Risk factors for neonatal sepsis include maternal factors, neonatal host factors, virulence of infecting organism and increase significantly the smaller and more immature the baby is. Maternal infection is one of the commonest identifiable reasons for spontaneous preterm labor. Thus, spontaneous vaginal delivery of a baby at less than 37 weeks is by definition a risk factor for neonatal sepsis. Risk factors for EOS are essentially those for GBS and include maternal intrapartum fever, prolonged rupture of membranes (PROM) more than 18 hours, prematurity less than 37 weeks and having a previous infant with GBS (Table 1). In the United States, widespread acceptance of intrapartum antibiotic prophylaxis (IAP) to reduce vertical transmission of GBS infections in high-risk women resulted in a significant decline in rates of GBS-related EOS [97]. However, despite the efforts to detect GBS colonization during pregnancy and provide appropriate GBS prophylaxis to colonized mothers, not all cases of early-onset GBS are prevented and GBS continues to be the most common cause of EOS in term neonates [39,40].

Overall, it is not believed that IAP has resulted in a change in pathogens associated with EOS; however, some studies among VLBW preterm infants have shown an increase in EOS caused by Escherichia coli [65]. Factors predisposing to LOS include being in intensive care unit, not receiving enteral feeds, not receiving maternal breast milk, having an indwelling catheter, or receiving total parenteral nutrition (TPN). Babies who had gut surgery or gut-related problems have a significant risk of nosocomial sepsis, probably related to being unable to feed enterally, with the development of unbalanced, potentially pathogenic, intestinal flora (Table 1).

In a previous multicenter, prospective cohort study aimed to assess the epidemiology of nosocomial infections in NICUs we found different patterns of risk factors between VLBW and heavier neonates [76]. Prolonged use of all antibiotics, but especially broad spectrum antibiotics, can result in an increased antibiotic resistance among normal commensal organisms and favors opportunistic transmission within the unit. Antibiotic therapy therefore may simply replace one pathogen for another pathogen, which itself may be more hazardous. Risk factors for Candida infection include BW <1500 g, TPN, presence of indwelling catheters, not receiving enteral feeds, mechanical ventilation, H2 receptor antagonists, abdominal surgery, peritoneal dialysis, exposure to broad spectrum antibiotics, especially third generation cephalosporins, and exposure to antenatal antibiotics.

CENTRAL LINE ASSOCIATED BLOODSTREAM INFECTIONS (CLABSI) AND CENTRAL LINE RELATED BLOODSTREAM INFECTIONS (CLRBSI)

Sepsis in neonates with vascular lines is a significant cause of morbidity and mortality in neonatal intensive care units. The incidence (expressed in relation to the duration, therefore in proportion to 1000 days of catheterization) is 3.9/1000 days in neonates with BW >2500 gr and11.3/1000 days in neonates with BW <1000 gr. The terms to describe bloodstream infections have often been used imprecisely and heterogeneously in the scientific literature, leading to confusing results (98). The so called Central Line Associated Blood-Stream Infections (CLABSI) are episodes of sepsis diagnosed within the first 48 hours after CVC insertion




or within 48 hours after its removal. In contrast, the term Central Line Related Blood-Stream Infections (CLRBSI) includes those episodes of sepsis with positivity of CVC blood cultures developing at least 2 hours before compared to peripheral blood cultures [99]. These infections increase hospital costs and length of stay, but have not generally been shown to independently increase mortality [100]. Thus, there is considerable interest in reducing the incidence of these infections to improve patients’ outcome and to reduce healthcare costs.

Four routes seem to have importance for the contamination of catheters: 1) Migration of skin organisms at the insertion site into the cutaneous catheter tract and along the surface of the catheter, with colonization of the catheter tip. This is the most frequent and recognized route of infection for short-term catheters 2) Direct contamination of the catheter hub by contact with not well washed hands or contaminated fluids 3) Less commonly, catheters might become hematogenously seeded from another focus of infection; 4) Rarely, infusate contamination leading to catheter related bloodstream sepsis (CRBSI) [101-103].

The most important pathogenic determinants of CRBSI are 1) Material of which the device is made up; 2) Host factors consisting of adhesion proteins, such as fibrin and fibronectin, which form a sheath around the catheter [104]; 3) Intrinsic virulence factors of the infecting organism, including the extracellular polymeric substance (EPS) produced by the adherent organisms [105].

Some catheter materials also have surface irregularities that enhance the microbial adherence of certain species (e.g., S. epidermidis and C. albicans) [106]. Silastic catheters are associated with higher risk of catheter infections than polyurethane catheters due to the formation of the fibrin sheath. C. albicans forms more easily biofilm on silicone elastomer catheter surfaces than polyurethane catheters. Additionally, certain catheter materials are more thrombogenic than others, predisposing to catheter colonization and infection. This association has led to emphasis on preventing catheter-related thrombus as an additional mechanism to reduce these infections. The most commonly reported causative pathogens remain coagulase-negative staphylococci, Staphylococcus aureus, enterococci, and Candida spp. Gram negative bacilli accounted for 19% and 21% of CLABSIs [107-110].

Clinical findings alone are not always reliable to establish the diagnosis of intravascular device–related infection because of their poor sensitivity and specificity. The most sensitive clinical finding, fever, has poor specificity. Moreover, in neonates fever is an inconstant symptom of infection. The presence of redness and purulence around the insertion site of the catheter has greater specificity but poor sensitivity Blood cultures positive for S. aureus, coagulase-negative staphylococci, or Candida species, in the absence of other identifiable sources of infection, should increase the suspicion for CRBSI. Improved symptomatology within 24 h after catheter removal suggests but does not prove that the catheter is the source of infection [79]. Clinical suspicion of infections and specific laboratory exams to establish the cause-effect relationship should be used to diagnose intravascular catheter-related infections.

The goal of an effective prevention program should be the elimination of CLABSI from all patient-care areas. Although this is challenging, programs have demonstrated success, but sustained elimination requires continued effort [111,112]. In our Department of Neonatology at the Bambino Gesù Children’s Hospital in Rome, an active surveillance intervention was conducted to improve the adherence to procedures recommended to reduce CLABSI. The intervention targeted clinicians’ use of the following issues: correct hand washing, full barrier precaution during insertion of the central line, cleaning the skin with chlorexidine in infants >1500 g, sterile manipulation of the catheter, reducing the number of manipulations of the catheter, prompt removal of unnecessary catheters. Several audits were made to reach all the medical and nurse staff. During a period of 21 months, direct surveillance of catheter insertion and manipulation was performed to check the adherence to infection control practices. Number of CLABSI and catheter days were collected from a hospital based infection control practitioner. From April 2011 to December 2012 the mean CLABSI rate, expressed as the number of infection episodes per 1000 catheter days, decreased from 14.9 to 35 (unpublished data). The number of bacterial CLABSI decreased significantly whereas the number of fungal CLABSI remained constant over that period of time. Thus, the adherence to bundles seems to be the tool to prevent infections from catheter insertion to catheter removal. These interventions have to be implemented to achieve the goal of zero CLABSI.

CLINICAL MANIFESTATIONS OF NEONATAL SEPSIS

Clinical signs of infection in the early stage of any infection in neonates may vary from mild to severe and usually are very subtle (Table 2). Variables affecting the manifestations include the duration of infection, virulence of the causative agent, and degree

Table 2: Symptoms and signs of neonatal sepsis.Temperature instability (rectal temperature ≤36°C or≥38°C)Decreases urinary output or anuria, serum creatinine >1.5 mg/dl, hypotensionImpaired peripheral perfusion (capillary refill time >2 sec)Apnea (cessation of breathing that lasts for at least 20 seconds or accompanied by bradycardia or oxygen desaturation)Increased oxygen requirement, increased requirement for ventilatory supportBradycardia, tachycardia, rhythm instabilityFeeding intolerance, abdominal distension, distended loops of the bowel, emesis, large prefeeding gastric residuals, diarrhea, gross blood in the stoolLethargy, hypotonia, irritabilitySkin lesions, petechial rash, scleremaWhite blood cells (WBC) count < 4 or > 20 x 109 cells/LImmature to total neutrophil ratio (I/T)> 0.2Platelet count < 100 x 109/LCRP >15 mg/LProcalcitonin ≥ 2 ng/mLGlucose intolerance(8-15 g/kg/day)>180 mg/dL or hypoglycemia (< 40 mg/dL)Acidosis, base excess (BE) <-10 mmol/L, lactate >2 mmol/L.




of maturity of host defence mechanisms. In preterm infants, symptoms are mostly unspecific. A baby may simply not be feeding well or be excessively sleepy, with a low level of alertness and some disturbance of peripheral perfusion. Tachypnoea, apnoea, or respiratory distress is the most common presenting signs of infection. A ventilated baby may require increased ventilatory support, having previously been stable or improving. Pneumonia is a possible diagnosis, but a number of other pathologies are also possible, including generalized sepsis, meningitis, cardiac disease and metabolic conditions with acidosis. Other symptoms may be cyanosis and shock. The temperature may be elevated, depressed, or normal. Fever in newborn infants may also be due to elevated environmental temperature, dehydration, or hematoma. A single temperature elevation is rarely associated with infection, but a sustained elevation is highly predictive. In premature infants with sepsis, subnormal temperatures and irregular fluctuations are observed as often as fever. Signs of fetal distress can be the earliest indication of infection in neonates with sepsis, beginning at or soon after delivery. Fetal tachycardia in the second stage of labor was evaluated as a sign of infection by Schiano and colleagues [113].

Early onset infection may be clinically indistinguishable from hypoxic ischemic encephalopathy at delivery. Progression from mild symptoms to death can occur in less than 24 h with certain organisms including GBS and Escherichia coli. GBS infection should be suspected in a baby who has more severe respiratory distress syndrome (RDS) than would be anticipated, as RDS and GBS pneumonia are radiologically indistinct. In severe cases persistent pulmonary hypertension of the neonate (PPHN), hypotension, metabolic acidemia, tachycardia and poor peripheral perfusion may develop and are poor prognostic factors. The nonspecific and subtle nature of the signs of sepsis in neonates is even more problematic in identifying sepsis in VLBW infants [114].

Septic infants can present with neurologic findings such as seizures and full fontanel even in the absence of meningitis. Gastrointestinal disturbances, including regurgitation, vomiting, large gastric residuals in infants fed with tube, and abdominal distention, are common and significant early signs of sepsis.

A variety of skin lesions accompany bacteremia, including abscesses, sclerema, petechia, etc. Severe sepsis may also lead to a septic shock, presenting with rapidly evolving symptoms, which reflect multiple organ failure, such as respiratory distress syndrome, secondary surfactant deficiency, pulmonary edema, pulmonary hypertension, oliguria, hypotension, metabolic acidosis, and disseminated intravascular coagulation (DIC).

DIAGNOSIS OF NEONATAL SEPSIS

Specific and non-specific diagnostic tests

Timely diagnosis of systemic infection in the neonate and prompt institution of antimicrobial therapy are essential in order to mitigate the high case fatality and to avert morbidity associated with it. The diagnosis is particularly difficult on the basis of clinical findings alone. The newborn infant responds similarly to a variety of stresses, regardless of their nature or location. The challenge is to differentiate infants who are truly septic, in whom

antimicrobial therapy could be life-saving, from those who have a low probability of infection, in whom antibiotics can be safely withheld or discontinued.

Among the laboratory tests indicative of bacterial sepsis the isolation of microorganisms from blood, urine, or cerebrospinal fluid remains the gold standard for definitive diagnosis. However, confirmation or exclusion of positive cultures requires days, and more importantly, the sensitivity of the culture methods is frequently low, due to the concomitant antibiotic therapy or to the combination of small blood sample volume and low colony counts. When a 0.5 mL blood sample is obtained for culture (a likely occurrence in NICUs), the probability to isolate organisms is 0.39 with one CFU/mL, 0.67 with two CFU/mL, 0.87 with four. A count of at least 4 CFU/mL and one ml blood volume are necessary to reach a probability of 0.98 [115]. Assessment of the concentrations of proteins, that are induced in response to sepsis, can serve as adjuncts to blood culture, to anticipate the time required for the positivization of microbiological cultures. Nonspecific laboratory investigations for the diagnosis of invasive bacterial infections remain the most important diagnostic aid for the management of septic neonates.

Total leukocyte count (>20000 or <5000), differential leukocyte count and morphology, total neutrophil count, total nonsegmented neutrophil count, neutrophil ratios, platelet count are the indices most commonly used. Contrary to older children and adults, the white blood cell (WBC) count does not accurately predict infection in neonates. A recent multicenter review of complete blood count (CBC) and blood cultures in neonates admitted to 293 neonatal intensive care units in the United States, showed that low WBC and absolute neutrophil counts, as well as immature to total neutrophil ratio (I:T ratio) were associated with increasing odds of infection (odds ratios 5.38, 6.84, and 7.90, respectively); however, the test sensitivities for detection of sepsis were low. Studies looking at serial values of WBC counts and I:T ratios have shown better outcomes: two serial normal I:T ratios and a negative blood culture in the first 24 hours of life had a negative predictive value of 100%, but the specificity and positive predictive value were 51% and 8.8%, respectively [69,116,117]. These haematological counts and ratios need to be interpreted cautiously and in conjunction with other clinical and laboratory parameters.

Further diagnostic tools are the acute phase reactants, endogenous peptides produced by the liver as part of an immediate response to infection or tissue injury. The most widely used acute phase reactant in neonates is CRP, a globulin that forms a precipitate when combined with the C-polysaccharide of Streptococcus pneumoniae. GA influences CRP kinetics, with preterm infants having a lower and shorter CRP response, compared with term healthy infants [118]. Given that there is a time lag of 6-8 hours following the stimulus, required for synthesis; the peak that is observed at 24 hours, and the half life that is 19 hours, clinicians usually add other tests besides CRP assessment. The specificity of CRP is low for EOS, as a number of prenatal conditions (maternal fever, fetal distress or stressful delivery, and vacuum delivery) may lead to its elevation in the absence of systemic infection and given the physiologic variation in CRP during the first days of life, all together limiting the use




of single CRP values [119,120]. On the other hand, quantitative CRP values, when repeated serially, are highly specific and have good sensitivity. In addition, serial measurements can be helpful in monitoring the response to treatment. Two serial CRP values <1 mg/dL, excluding the investigation soon after birth, carry a 99% negative predictive value. Recent studies, using CRP cut off values of 1.2–6 mg/dl to diagnose sepsis and guide duration of therapy in EOS and LOS, showed specificity between 84–96% and a negative predictive value range of 93-99%.The clinical practice of using higher CRP cut off values led to fewer days of antibiotics without an evidence of infection relapse [121].

Microerythrocyte sedimentation rate (micro-ESR) by the use of a microhematocrit tube has been developed over 50 years ago and is considered of little value in either diagnosing or monitoring infection in the neonate. Normal values change significantly during the first two weeks of life, and sensitivity is low due to the rise delay and to the long time required for normalization after clinical recovery.

Procalcitonin, a propeptide of calcitonin, is an acute phase reactant produced by monocytes and hepatocytes. It is released into the blood 3 to 6 hours after endotoxin injection and increases up to 24 hours, more rapidly than CRP. The increase with infection makes PCT a potential marker for early detection of sepsis. However, daily variations of PCT also in uninfected neonates have been reported, with a peak on day 1-2 of life, followed by a regular decrease to normal values [122]. This physiological increase could partially limit the usefulness of PCT in the diagnosis of EOS.

We have confirmed these data measuring PCT levels in critically ill but uninfected patients admitted to NICU in the first three days of life. We considered 800 uninfected neonates on admission to the ward (GA 34+4 W, BW 2130+863g); 229 of them were VLBW (29+3W, BW 1123+258g). The median values of PCT in all neonates were: 0.40 ng/ml; IQR 0.21-1.20) in the first, 2.38 ng/ml (IQR 0.77-8.76) in the second and 0.52 ng/ml (IQR 0.28-1.37) in the third day. In the group of 229 uninfected VLBW infants, the median PCT values were: 0.35 ng/ml (IQR 0.21-0.77) in the first, 4.23 ng/ml (IQR 0.65-11.23) in the second and 0.51 ng/ml (IQR 0.35-1.40) in the third day of life (data unpublished).

In a multicentric study on the usefulness of PCT in differentiating septic with LOS from non-septic infants [123], we recommended a PCT cut off value of >2.4 ng/ml as the most accurate level for differentiation of sepsis in neonates, regardless of gestational age, with a sensitivity of 62% and a specificity of 84%. This cut off value exceeds the physiologic increased PCT median value of 2.38 ng/ml, observed in uninfected infants in the second day of life (data unpublished), perhaps improving the performance of PCT also in detecting EOS. In our opinion, PCT should be used as an adjunct, rather than a replacement, to CRP to improve the diagnostic accuracy. Moreover, it should be measured in sequence to CRP rather than simultaneously. In fact, if PCT is measured simultaneously with CRP sensitivity is reduced, as they are two independent tests, and overall sensitivity is the product of individual sensitivity of the two tests. But if the two tests are used in sequence, i.e. PCT is measured only in patients with negative CRP, sensitivity increases since the probability of PCT is conditioned by the negative result of the

first CRP test. So, sensitivity of PCT increases if measured only in patients with negative CRP. Increases of the above indices have been also associated with several non-infectious perinatal events causing tissue injury or inflammation, such as maternal hypertension, mode of delivery, asphyxia, respiratory distress syndrome, brain hemorrhage, and meconium aspiration. In these situations, the use of CRP and subsequently the measure of PCT in patients with negative CRP negative can improve the ability to diagnose an infectious episode. Further studies are needed to better clarify the use of PCT in clinical practice.

Helpfulness of cytokine profile in the diagnosis of neonatal infection

The time required for blood cultures and for hematological/biochemical tests indicative of infection to become positive limits their usefulness in the early diagnosis of sepsis in newborns. Furthermore, infants (and even more preterm neonates) at the beginning of the infectious episode may not manifest changes in conventional biochemical markers, as is the case for the CRP. Moreover, the prompt diagnosis is crucial in newborns, since even a small delay in the beginning of an antibiotic treatment may adversely and irreversibly affect the outcome of the infection.

Neonates react to bacterial sepsis with an exaggerated inflammatory response, largely mediated by cytokines. Therefore, the current diagnostic research is considering how they can be used in early diagnosis of neonatal sepsis [124,125]. The inflammatory cascade induced by a bacterial infection begins with the activation of macrophages and the release of cytokines (tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1 β), interleukin-6 (IL-6) and growth factors interleukin-3 (IL-3), granulocyte colony stimulating factor (G-CSF). These chemical mediators trigger the acute phase reactants synthesis by the liver and the activation of neutrophils. The increase of cytokines levels occurs much earlier than the one of conventional hematological and biochemical markers of infections (Figure 1).

Several cytokines and receptors have been studied for the early diagnosis of neonatal sepsis, such as IL-6, IL-8, IL-10 and TNF- α. IL-6 and IL-8 serum levels increase very rapidly at the onset of a bacterial infection, but promptly normalize within 24 hours, limiting their ability to be used in a clinical setting. In particular, the short half-life of IL-6 is caused by its binding to plasma proteins, early hepatic clearance, and inhibition by other cytokines.

TNF-α has not shown to have high sensitivity. Plasma adenosine, an endogenous plasma metabolite that rises with hypoxia and stress, is elevated in newborn blood plasma and inhibits production of TNF-α by monocytes with preservation of IL-6 synthesis [126]. This action may contribute to neonatal susceptibility to infection with intracellular pathogens. Larger studies are necessary to evaluate if a combination of cytokine profiles may increase the likelihood of identifying infection more than single measurements.

Neutrophil CD64 and neutrophil/monocyte CD11B

Recent improvements in the quantitative flow cytometry has made possible a new diagnostic approach, concerning the identification of membrane antigens expressed on blood cell surfaces, when cells are activated by an infectious episode.




Compared to the other conventional immunoassays, quantitative flow cytometry allows the detection of cells activation surface markers in a short time, with little blood volumes [127]. Furthemore, the concentration of circulating cytokines may not necessarily reflect their biologic activity. The detection of the cellular response to cytokines may be a better way to identify an immune response to a bacterial invasion. This applies to both antigens expressed on the membranes of neutrophils and lymphocytes.

CD64 is an antigen normally expressed at low levels on the surface of neutrophils, both in term and in preterm VLBW infants. Its expression increases after a bacterial infection, thus suggesting being an effective marker in the diagnosis of LOS in preterm infants. Genel and colleagues showed that CD64 had a sensitivity and specificity to identify neonatal sepsis of 81% and 77% respectively, with a negative predictive value of 75% [128]. The same effect would exist even in the context of EOS in term infants. The accuracy of this parameter increases if analyzed by PCR.

Also the expression of other antigens, such as CD66b and CD33, seems particularly pronounced on the surface of neutrophils during sepsis. These antigens, in combination with the increased expression of lymphocyte antigen CD19, may be predictive of sepsis. Also CD11b is an antigen normally expressed at low concentrations on the surface of neutrophils [129]. Its expression increases, considerably and within few minutes, when inflammatory cells go into contact with bacteria and endotoxins [130]. This makes CD11b particularly “powerful” (sensitivity 66% and specificity 71%) in predicting a bacterial infection.

While the presence of CD11b appears to be particularly sensitive and specific to diagnose early infections of neonates with GA >28 weeks, data are more divergent concerning late infections in preterm infants. As shown, the detection of activated neutrophils surface antigens CD64 and CD11b by quantitative flow cytometry appears to be particularly promising. Their concentration increases regardless of the immaturity of the immune system of the newborn, and such technique allows studying more parameters at the same time, in a short time and by using small volumes of blood. Costs and processing time could be barriers to the use of these markers in clinical practice. Nonetheless, it might be interesting to compare CD64 and CD11b with CRP and to determine the cut–off for each parameter. This implies the need to perform large studies among infected neonates.

SCREENING PANELSExcluding cultures, none of the conventional biochemical

and/or immunological tests, when used alone, is currently considered sufficiently sensitive and specific to reliably diagnose sepsis in neonates. Thus, clinicians have considered some screening panels, i.e. a combination of exams, to increase the sensitivity and the specificity to confirm or exclude the infection. To date, the best combination of markers to detect neonatal sepsis consists of performing IL-6 and IL-1β one-two days before the onset of symptoms, IL-6 (or IL-1β, IL-8, CD11b, CD64, GCSF, TNF-α), CRP, procalcitonin, and haematological indices on day 0, and CRP and haematological indices on the following days to

monitor the response to therapy. Overall, the negative predictive value is remarkably good, approaching 100% in some studies. Due to their high negative predictive value, screening panels may produce a significant decrease in the use of antimicrobial agents [131].

However, although laboratory tests can be used to improve the accuracy of the clinical evaluation, clinical signs of sepsis remain the most important criteria for the use of antimicrobial agents and if the history or physical examination of the baby conflicts with negative exams antimicrobial therapy should be started.

MOLECULAR BIOLOGY IN THE EARLY DIAGNOSIS OF NEONATAL SEPSIS

Blood culture is the current gold standard for the detection of sepsis, since bacteria isolated from the blood can be isolated to identify species and antibiotic susceptibility. Nonetheless, the value of blood cultures is impaired by the delay in the timing of results and by the fact that cultures become positive only in 30% of patients. Important advances in the development of sepsis-specific biomarkers have been made with molecular diagnostics, studies of real-time PCR and a broad range of conventional PCR assays.

There are many molecular-based approaches for diagnosis of neonatal infection such as:

1) whole blood directly tested by target amplification;

2) whole blood pre-enrichment before target amplification;

3) fluids from positive blood culture bottles tested by polymerase chain reaction (PCR);

4) Nucleic Acid Sequence-Based Amplification (NASBA);

5) Nucleic Acid Amplification Tests (NAAT);

6) PCR in conjunction with sequencing or microarray analysis;

7) non amplification-based fluorescence in situ hybridization (FISH).

Currently, these techniques should be seen as adjunctive methods in the diagnosis of neonatal sepsis, with limitations that include the inability to provide information about antibiotic susceptibility, as well as significant cost implementation in clinical practice.

PROTEOMICS AND GENOMICSSince the pathophysiology of sepsis may involve all cells and

tissues, at least 180 distinct molecules have been suggested as potential biological markers of sepsis. However, only 20% of them have been assessed with appropriate studies for their possible use in clinical setting, with current diagnostic uncertainty.

Modern applications of molecular pathology embrace various disciplines: genomics (sequence DNA), transcriptomics (mRNA identification), proteomics (protein identification) and pharmacogenomics (genes that define the behavior of drugs). Proteomics is the study of expressed proteins in a tissue, cell, or organism at a given moment. It is more clinically significant




and easier to translate into diagnostic tools and therapeutical strategies than genomics, which studies DNA. In fact, proteomics directly examines functional molecules and not the source code. Evidence to support this statement is that protein quantity and activity do not show a relationship with mRNA amount. Proteomics has been applied to the search for biomarkers and production of protein profiles that can rapidly help the prediction, early diagnosis, and treatment of human diseases [132,133]. Several information can be obtained from proteomics study, although only parts of these are biologically significant. To analyze proteomics data from SELDI-TOF (enhanced laser desorption/ionization time of flight) outputs, Buhimschi et al., developed a strategy to extract significant proteomic biomarkers characteristic for intra-amniotic inflammation, based on sequentially applied filter preferences. This strategy was called mass restricted (MR) scoring. The MR score indicates the number of identifiable markers among the following factors of innate immunity: neutrophil defensin-2, neutrophil defensin-1, S100A12 (calgranulin C) and S100A8 (calgranulin A) [134,135]. An MR score of 3-4 indicated the presence of inflammation, while a score of 0-2 excluded it. The same authors prospectively validated the clinical utility of the MR score in predicting preterm births and neonatal sepsis in a cohort of 169 consecutive women with single pregnancies. An MR score of 3-4 had the highest accuracy (92.6%) in diagnosing intra-amniotic inflammation, and was significantly better than white blood cell (WBC) count or IL-6. Therefore, high MR scores are associated with preterm delivery, histological chorioamnionitis, and EOS. Detection of monomeric S100A8 (calgranulin A) in the amniotic fluid was closely associated with EOS. Both GA and the presence of S100A12 and S100A8 in amniotic fluid tightly correlated with neonatal neurodevelopmental impairment. In conclusion, the amniotic fluid proteome closely reflects the fetal inflammatory response to intra-amniotic infection and SELDI-TOF technology carries the attributes to allow a direct, rapid and reliable identification of proteomic biomarkers of intrauterine inflammation. The sensitivity and specificity of real-time PCR assay was 96.2% and 100% respectively, 35 with a limit of recognition for E.Coli and group B Streptococcus, Makhoul et al., showed that sensitivity, specificity, positive predictive value and negative predictive value of Staphylococcus-specific PCR, used for detection of staphylococcal bacteremia, was 57.1%, 94.7%, 53.3% and 95.4%, respectively [136]. A technique based on PCR to amplify the gene cfb directly from swabs to accurately identify the colonization by group B streptococcus, with results within 1-2 hours, was recently approved for use in both prepartum and intrapartum by the Food and Drug Administration (FDA), while NAAT for Perinatal Group B Streptococcus identification has been considered in the most recent Guidelines from CDC [8]. Despite the cfb-PCR has been approved by the FDA, Atkins showed, by comparing the PCR and double (direct inoculation on selective agar and broth) culture methods in a study of 233 samples, that PCR had sensitivity, specificity, positive and negative predictive value of 86.8%, 95.2%, 88.1% and 94.6%, respectively, with a false negative rate of 13.2% [137]. Using whole blood directly in a target amplification-based assay for detecting bacteria has the advantage of rapid diagnosis, but the challenges of suboptimal sensitivity and specificity. Several strategies are under investigations to improve this problem, as an increase of

blood volume (but it is not always possible, particularly in small infants), improvement of extraction procedures to obtain greater recovery of bacterial nucleic acid over human genomic DNA, or whole blood pre-enrichment. Bacterial detection platforms such as DNA microarray, FISH, and mass spectrometry do not require target amplification. Several studies reported that the sensitivity and the specificity of these molecular-based approaches were from 98-100% and from 99-100%, respectively. The major benefit of a molecular test is speed; however, so far, none of the rapid tests has been shown to have a sensitivity and specificity sufficient to replace standard blood culture techniques that carries the additional important advantage of the ability of testing pathogens antibiotics susceptibility. The possibility of understanding the genetic contribution to response to microbial pathogens remains one of the most stimulating prospects of the unravelling of the human genome. The identification of strong associations between certain genetic polymorphisms and susceptibility to severe sepsis supports further research using appropriate association studies [138]. Recent evidence that the genetic background of the host affects the systemic response to infection has stimulated considerable interest in the evaluation of genetic susceptibility to sepsis, concerning in particular factors of the initial immune response of the innate immunity, as TLRs, MBL, nucleotide-binding oligomerization domain (NODs) and cytokines [139-141]. Genes involved in the regulation of immune function, particularly the systemic inflammatory response, have been evaluated in an attempt to identify markers of infection in neonates [142]. A research performed on two polymorphisms of TNF did not provide evidence that these can influence the incidence of early-onset sepsis in premature infants [19]. Other studies about IL-6 failed to identify a strong genetic correlation [142,143]. A recent metanalysis assessed the evidence for the association of the IL-6 (-174C) polymorphism (guanidine to cytosine transition at position -174 nucleotides relative to the transcription start site in the interleukin-6 gene) with the risk of sepsis in Very Light Birth Weight (VLBW) newborn infants. The results of six cohort studies including a total of 1323 VLBW infants found no significant association between the IL-6 (-174C) polymorphism and sepsis. These data did not support screening infants for this allele in order to guide selective antimicrobial prophylaxis [144]. Del Vecchio et al., compared the prevalence of TNF-á and IL-10 polymorphisms in preterm neonates with late-onset sepsis with a noninfected reference group. In the septic neonates, 308G-TNF-á and 1082-IL-10 polymorphisms resulted, in homozygous and heterozygous forms, more frequent with statistical significance. The authors, however, concluded that the analysis of a larger group of subjects was needed to confirm these data [145]. Recently, a significant association of IL-6-174CC and IL-10-1082GG genetic polymorphisms in homozygosis with increased risk of mortality was reported. Moreover, the studied genotypes were significantly higher in neonates who required inotropic support and those who developed disseminated intravascular coagulopathy.

On the whole, these preliminary studies tend to confirm that genetic variations and single nucleotide polymorphisms of factors involved in the immune defense against infection are significantly involved in the pathogenesis of sepsis, and may influence the outcome. Data, however, are still insufficient to




consider these factors useful for the evaluation of the risk to develop sepsis, to include any of these markers in the screening panels currently used for sepsis early diagnosis, or to guide the treatment of infection in the neonate.

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Ronchetti MP, Bersani I, Piersigilli F, Auriti C (2017) Neonatal Sepsis Arch Paediatr Dev Pathol 1(3): 1015.

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