Innate Immune System

The innate immune system produces an immediate immunologic response and is capable of doing this without previous exposure to a specific pathogen. Recognition of pathogens occurs by identification of conserved biologic regions known as pathogen-associated molecular patterns (PAMPs). Recognition receptors, such as TOLL-like receptors, NOD-like receptors and RIG-like receptors, identify and respond to PAMPs with the production of cytokines and proinflammatory responses that activate the adaptive immune system. Studies comparing neonatal and adult innate immune functions show that neonatal cells have a decreased ability to produce inflammatory cytokines, especially tumor necrosis factor (TNF) and interleukin (IL)-6. In addition, they induce IL-10 production, which in itself is capable of inhibiting synthesis of proinflammatory cytokines. Neutrophil and dendritic cell functions are also reduced; neutrophils show a decreased expression of adhesion molecules, as well as a decreased response to chemotactic factors, and dendritic cells have a decreased capacity of producing IL-12 and interferon (IFN) gamma. The overall reduction in cytokine production in neonates also results in decreased activation of natural killer cells. Impairment of the innate immune system leads to an increased susceptibility to bacterial and viral infection in this population.

Adaptive Immune System

The adaptive branch of the immune system is designed to eliminate specific pathogens. In newborns, the adaptive immune system slowly increases its function toward an adultlike response, minimizing the otherwise overwhelming inflammatory response that would occur 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. For example, the reduction of cell-mediated immunity increases the risks of infections caused by intracellular pathogens, such as Listeria , Salmonella , herpes simplex virus (HSV), cytomegalovirus, and enteroviruses.

Transplacental passage of maternal immunoglobulin G (IgG) is inversely related to gestational age and limits the functional ability of the neonate to respond to certain pathogens. Minimal IgG is transported to the fetus in the first trimester, whereas fetal IgG rises in the second trimester from approximately 10% at 17 to 22 weeks’ gestation to 50% at 28 to 32 weeks’ gestation. Thus, preterm infants lack adequate humoral protection against a number of infant 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 the infant’s susceptibility to encapsulated bacterial infections ( Streptococcus pneumoniae , Haemophilus influenzae , Neisseria meningitidis ). 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.

Complement

Complement levels increase with increasing gestational age, 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.

Group B-streptococcus

Despite widespread use of IAP to prevent vertical transmission of invasive GBS disease, missed opportunities for prevention exist and GBS remains the most common organism associated with EOS in the United States. 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. GBS are gram-positive encapsulated bacteria for which 10 different serotypes have been identified; serotype III strains are responsible for most of disease (54%). 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. GBS possess different virulence factors that determine its ability to cause invasive disease: (1) capsular polysaccharide, which helps evade phagocytosis; (2) pili, which allows adherence of GBS to the host’s epithelial cells as well as transepithelial migration; and (3) C5a peptidase, which inhibits human C5a, a neutrophil chemoattractant 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, with mortality of 20% to 30% among infected infants of less than 33 weeks’ gestation, compared with a mortality of 2% to 3% in full-term infants.

Escherichia coli

E 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. 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 and meningitis, as well as with increased risk of mortality when compared with K1-negative strains. Some studies suggest a more aggressive presentation for infants infected with E coli , with a higher risk of thrombocytopenia and death in the first days of life. Several US studies have shown high rates of ampicillin resistance in E coli strains that infect newborns. Although some studies have shown an association between intrapartum antibiotic exposure and ampicillin-resistant E coli , ampicillin resistance has increased throughout the community and a direct link between intrapartum use of ampicillin and the higher likelihood of resistance has not been established.

Listeria monocytogenes

Listeria is a facultative anaerobic, gram-positive bacterium found in soil, decaying vegetation, fecal flora, and raw unprocessed food. 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 ActA, phospholipase C, and lecithinase allow polymerization of actin and lysis of phagosomal membranes, enabling cell-to-cell transmission. Pregnant women have 17% higher risk of Listeria infection than nonpregnant women, and infection has been associated with spontaneous abortions and stillbirths. Early neonatal infections have a similar clinical presentation, as EO GBS infections, 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. Suspicion for Listeria sepsis should be increased in ill infants of mothers who have consumed raw milk, unpasteurized cheeses, or other unprocessed food products that have been contaminated with the organism.

Other Bacterial Etiologic Agents Seen in EOS

Other less common but important pathogens associated with EOS include other streptococci ( Streptococcus pyogenes , viridans group streptococci, S pneumoniae ), enterococci, staphylococci, and nontypable H influenzae . S pyogenes (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. 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. 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. Although nontypable H influenzae frequently colonizes the maternal genital tract, neonatal infection is relatively rare, but with high mortality rates, especially in preterm neonates. 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.

CoNS and Staphylococcus aureus

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. S aureus is associated with 4% to 8%. 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. Methicillin-resistant S 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 S aureus .

Gram-negative Organisms

Gram-negative organisms are associated with about one-third of cases of LOS, 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 GI tract, contamination of total parenteral nutrition or formulas, and bladder catheterization devices. The most common gram-negative organisms isolated include E coli , Klebsiella , Pseudomonas , Enterobacter , Citrobacter, and Serratia . In some case series, Klebsiella is recognized as the most common gram-negative agent associated with LOS, ranging from 20% to 31% of cases. Infections caused by Pseudomonas have been associated with the highest mortality. 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 (CNS) infections and abscesses.

Candida Infections

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. Candida albicans and Candida parapsilosis are the species most commonly associated with disease in neonates. 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. 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 (CSF) culture, as many as 50% will have a negative blood culture; the discordance of blood and CSF cultures underscores the need for a lumbar puncture (LP). Prompt removal of contaminated catheters is also 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.

HSV

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. For a detailed discussion on neonatal HSV infection, refer to the article by Kimberlin and colleagues, elsewhere in this issue.

General Evaluation of Neonatal Sepsis

A neonate with signs and symptoms of sepsis (see Table 3 ) requires prompt evaluation and initiation of antibiotic therapy. Blood, CSF (as clinical condition allows), and urine cultures (useful only after the third day of life) should be obtained. Chest radiograph is indicated in patients having respiratory symptoms. If disseminated herpes is suspected (herpetic skin lesions, elevated hepatic transaminases, maternal peripartum herpes infection), surface cultures from conjunctiva, mouth, skin, and anus, as well as herpes DNA polymerase chain reaction (PCR) from CSF and blood should also be ordered. Ancillary tests, such as complete blood count (CBC) and C-reactive protein (CRP), should not preclude a sepsis evaluation in a neonate, because they can be normal (see the following sections). If positive, however, they can be useful in supporting the diagnosis and determining length of therapy. Careful maternal and exposure history targeted toward identifying potential risk factors (see Table 1 ), as well as a complete physical examination, including skin and catheter insertion sites, should be obtained.

Complete Blood Cell Count

Contrary to older children and adults, the white blood cell (WBC) count does not accurately predict infection in neonates. A recent multicenter review of CBCs and blood cultures in neonates admitted to 293 neonatal intensive care units (NICUs) in the United States, showed that low WBC and absolute neutrophil counts, as well as high 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. Murphy and Weiner, in a single-center historical cohort study, showed that 2 serial normal I:T ratios and a negative blood culture in the first 24 hours of life had a negative predictive value (NPV) of 100% (95% confidence interval [CI]: 99.905%–100%), but the specificity and positive predictive value were 51.0% and 8.8% respectively. CBC values need to be interpreted cautiously and in conjunction with other clinical and laboratory parameters.

CRP

CRP was first described in the 1930s and since then multiple studies have shown elevation of the CRP in several infectious and noninfectious etiologies that share a common background of inflammation or tissue injury. In neonates, serial measurements of the CRP in the first 24 to 48 hours of symptoms increases the sensitivity of the test, with suggestion that normal CRP values during this period have a 99% negative predicted value for determination of infection. In contrast, elevated levels of CRP may be more difficult to interpret, especially for diagnosis of EOS, because factors such as premature rupture of membranes (PROM), maternal fever, pregnancy-induced hypertension, prenatal steroid use, and fetal distress may also cause elevation of the CRP. Additionally, studies have suggested a physiologic variation of the CRP during the first few days of life limiting the use of single values. Gestational age influences CRP kinetics, with preterm infants having a lower and shorter CRP response compared with healthy term infants. Studies suggest that CRP is best used as part of a group of ancillary diagnostic tests to help determine if an infant has infection, rather than as a single test.

Procalcitonin

Tissue release of procalcitonin (PCT) increases with infection, making it a potential marker for early detection of sepsis. PCT differs from CRP, in that PCT levels increase more rapidly and may be more useful for detection of EOS. Auriti and colleagues, in a multicenter, prospective observational study of 762 neonates, showed a significant increase in the median value of PCT level in neonates with sepsis compared with those without sepsis (3.58 vs 0.49 ng/mL; P <.001), In addition, a cutoff value of 2.4 ng/mL was suggested 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%. A meta-analysis of 16 studies (1959 neonates), showed that PCT had a pooled sensitivity and specificity of 81% and 79% respectively. Although these are promising results, further studies are needed to clarify the use of PCT in clinical practice.

Mannose-Binding Lectin

Mannose-binding lectin (MBL) is a plasma protein, primarily produced by the liver, with an important role in the innate immune defense. MBL activates the lectin pathway of the complement system, increasing opsonization and enhancing phagocytosis. Genetic polymorphisms in the MBL gene have been associated with an increased risk of sepsis. In a recent study in which MBL levels were measured in 93 neonates, development of sepsis was associated with lower levels of MBL and with the presence of BB genotype in exon 1 of the MBL gene. MBL remains a research tool with further studies needed to confirm diagnostic utility.

Cytokine Profile

Multiple cytokines have been studied for diagnosis of neonatal sepsis including IL-6, IL-8, IL-10, and TNF-alpha. IL-6 and IL-8 increase very rapidly with bacterial invasion, but they promptly normalize in serum levels (within the first 24 hours), limiting their ability to be used as clinical markers. TNF-alpha has not shown to have high sensitivity, but the ratio of IL-10 and TNF-alpha has been used for diagnosis of LOS in VLBW neonates with some success. Evaluating a combination of cytokine profiles may increase the likelihood of identifying infection more than single measurements.

Neutrophil CD64 and Neutrophil/Monocyte CD11B

The specific markers, neutrophil CD64 and neutrophil/monocyte CD11B, are cell surface antigens whose production increases after activation of leukocytes by bacteria and therefore can potentially be used for diagnosis of neonatal sepsis. Their upregulation precedes that of CRP, suggesting potential use in EOS. A recent study by Genel and colleagues showed that CD64 had a sensitivity and specificity to accurately identify neonatal sepsis of 81% and 77% respectively, with an NPV of 75%. Similarly, CD11b had a sensitivity and specificity of 66% and 71%. Cost and processing time may be barriers to use of these markers in clinical practice.

Molecular Techniques for Early Detection of Neonatal Sepsis

Important advances have been made in molecular diagnostics, and studies of real-time PCR and a broad range of conventional PCR assays suggest improved sensitivity and specificity for sepsis diagnosis. A meta-analysis done by Pammi found that sensitivity and specificity of real-time PCR was 0.96 (95% CI for sensitivity: 0.65–1.00 and 95% CI for specificity: 0.92–0.98). Similarly, broad-range PCR had a sensitivity of 0.95 (95% CI 0.84–0.98) and specificity of 0.98 (0.95–1.0); however, neither test achieved the minimum limits of sensitivity or specificity set up by the study and results were insufficient to replace blood cultures for diagnosis of neonatal sepsis. 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 of implementation in clinical practice. An exception to this is the use of HSV PCR for the diagnosis of HSV encephalitis, which is the gold standard test for this condition. The role of HSV PCR from the blood in diagnosing disseminated HSV infection is less clear.

Genomics and Proteomics

Exciting alternatives for detection of neonatal sepsis include the use of genomics and proteomics for identification of host response biomarkers. Genomics targets genes that are upregulated with infection and proteomics analyzes the structure, function, and interactions of proteins produced by a particular gene. Early studies in neonates have suggested potential utility in these techniques for identification of sepsis and necrotizing enterocolitis. A score based on proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid, was used to withhold antibiotics in 45% of patients with suspected infection and to discontinue antibiotics in 16%. Studies are needed for validation of this score, as well as for detection of other potential biomarkers.