**1. Introduction**

58 Neonatal Bacterial Infection

2004;89(3):F272-3-F-3.

[49] Pourcyrous M, Bada HS, Korones SB, Barrett FF, Jennings W, Lockey T. Acute phase reactants in neonatal bacterial infection. Journal of Perinatology: Official Journal of the

[50] Turner MA, Power S, Emmerson AJB. Gestational age and the C reactive protein response. Archives of Disease in Childhood Fetal and Neonatal Edition.

[51] Doellner H, Arntzen KJ, Haereid PE, Aag S, Austgulen R. Interleukin-6 concentrations

[52] Chiesa C, Osborn JF, Pacifico L, Natale F, De Curtis M. Gestational- and age-specific CRP reference intervals in the newborn. Clinica Chimica Acta; International Journal of

[53] Ng PC. Diagnostic markers of infection in neonates. Archives of Disease in Childhood

[54] Malik A, Hui CPS, Pennie RA, Kirpalani H. Beyond the complete blood cell count and C-reactive protein: a systematic review of modern diagnostic tests for neonatal sepsis.

[55] Dandona P, Nix D, Wilson MF, Aljada A, Love J, Assicot M, et al. Procalcitonin increase after endotoxin injection in normal subjects. The Journal of Clinical Endocrinology and

[56] Vouloumanou EK, Plessa E, Karageorgopoulos DE, Mantadakis E, Falagas ME. Serum procalcitonin as a diagnostic marker for neonatal sepsis: a systematic review and meta-

[57] Lam HS, Ng PC. Biochemical markers of neonatal sepsis. Pathology. 2008;40(2):141-8. [58] Simms HH, D'Amico R. Lipopolysaccharide induces intracytoplasmic migration of the polymorphonuclear leukocyte CD11b/CD18 receptor. Shock (Augusta, Ga).

[59] Lehr HA, Krombach F, Münzing S, Bodlaj R, Glaubitt SI, Seiffge D, et al. In vitro effects of oxidized low density lipoprotein on CD11b/CD18 and L-selectin presentation on neutrophils and monocytes with relevance for the in vivo situation. The American

[60] Ng PC, Li G, Chui KM, Chu WCW, Li K, Wong RPO, et al. Neutrophil CD64 is a sensitive diagnostic marker for early-onset neonatal infection. Pediatric Research.

[61] Haque KN. Neonatal Sepsis in the Very Low Birth Weight Preterm Infants: Part 2: Review of Definition, Diagnosis and Management. Journal of Medical Sciences.

[62] Van Lente F, Pippenger CE. The pediatric acute care laboratory. Pediatric Clinics of

[63] Chiesa C, Panero A, Osborn JF, Simonetti AF, Pacifico L. Diagnosis of neonatal sepsis: a

[64] Kukkonen AK, Virtanen M, Järvenpää AL, Pokela ML, Ikonen S, Fellman V. Randomized trial comparing natural and synthetic surfactant: increased infection rate after natural surfactant? Acta Paediatrica (Oslo, Norway: 1992). 2000;89(5):556-61.

clinical and laboratory challenge. Clinical Chemistry. 2004;50(2):279-87.

in neonates evaluated for sepsis. The Journal of Pediatrics. 1998;132(2):295-9.

California Perinatal Association. 1991;11(4):319-25.

Clinical Chemistry. 2011;412(19-20):1889-90.

Metabolism. 1994;79(6):1605-8.

1995;3(3):196-203.

2004;56(5):796-803.

2010;1(3):11-7.

Fetal and Neonatal Edition. 2004;89(3):F229-35-F-35.

analysis. Intensive Care Medicine. 2011;37(5):747-62.

Journal of Pathology. 1995;146(1):218-27.

North America. 1987;34(1):231-46.

Archives of Pediatrics & Adolescent Medicine. 2003;157(6):511-6.

Although diagnostic and therapeutic approaches to neonatal sepsis considerably progressed over the last decades, distinguishing infected from non-infected patients still remains a major challenge, especially in the early phase of disease when symptoms are often subtle and unspecific. Development and application of potent antibiotic medication and the advances in neonatal care could improve treatment but incidence of neonatal sepsis is still high. Compared to an incidence rate ranging from 1.5 to 3.5 per 1000 for neonatal early onset sepsis (EOS) and up to 6 per 1000 live births for late onset sepsis (LOS) in developed countries, the reported incidence of neonatal sepsis varies from 7.1 to 38 per 1000 live births in Asia and from 6.5 to 23 per 1000 live births in Africa (Vergnano et al., 2005). The physiologic immature state of the immune system and reduced levels of preformed maternal antibodies in preterm infants together with organ immaturity and a lower expression of major histocompatibility complex (MHC) class II antigens on monocytes contribute to a disturbed equilibrium of proand anti-inflammatory factors resulting in a reduced immune defence making the preterm infant more susceptible for sepsis and its short and long term complications (Azizia et al., 2012; Stoll et al., 2004). Prospective data collection of 16 participating centers of the National Institute of Child Health and Human Development Neonatal Research Network revealed a declining incidence of blood culture proven EOS from 19.3 per 1000 in 1991-93 to 15.4 per 1000 live births in 1998-2000 among very low birth weight (VLBW) infants, whereas the incidence of late-onset septicemia was 22% and remained essentially unchanged over the observed period of time. However, the potentially life threatening character of EOS is reflected by high mortality rates reaching between 1.6% in nonblack term infants and 37% in preterm infants with VLBW (Fanaroff et al., 2007; Weston et al., 2011). Infants in this study without EOS showed a significantly reduced mortality risk of 13%.

© 2013 Cimenti et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Cimenti et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Considering these data it is comprehensible that many neonatologists hazard the consequences of possibly unnecessary exposure to antimicrobial agents in neonates suspected for sepsis with unspecific symptoms and uncertain infectious state to avoid fatal outcome caused by a delay in treatment. The above-mentioned declined incidence of EOS was mainly caused by a change in the pathogen distribution showing a decline in group B streptococcus (GBS) sepsis but an increase in Escherichia coli sepsis with a rate of 85% of ampicillin resistance (Fanaroff et al., 2007). As the use of broad-spectrum antimicrobial agents like the combination of ampicillin and an aminoglycoside is considered as the optimal treatment of infants with suspected EOS (Polin, 2012), the progressively increasing burden of antimicrobial resistance would actually require a more targeted drug therapy in the future to confine human and economic costs. The often unspecific early symptoms and the potential rapid deterioration necessitate early identification of patients at risk. This should help to avoid a delay in treatment and could prevent further complications. On the other hand, overtreatment of newborn infants with maternal risk factors and uncertain infectious status suspected for sepsis could also be reduced helping to avoid prescription of unnecessary prophylactic broad-spectrum antibiotic medication, to restrain the development of antimicrobial resistance, exposing the patients to possible severe adverse effects and help to increase cost effectiveness.

The Role of Immature Granulocyte Count

and Immature Myeloid Information in the Diagnosis of Neonatal Sepsis 61

results are delayed and can normally be expected within three days, whereas in infants up to an age of 72 hours blood cultures require a longer incubation time. In a retrospective observational study comprising more than 2900 neonates the median time to positivity of blood cultures was significantly shorter in Gram negative (11.2 hours; 8.5-15.7) compared to Gram positive organisms (23.6 hours; 15.3-4.6). These findings could have important clinical implications to optimize antimicrobial therapy. The authors suggest targeting only for Gram positive germs when the blood culture is still negative after 48 hours and to cease treatment in well-being infants without clinical and laboratory signs of infection after 72 hours when

Generally, blood culture-proven EOS has been described as quite uncommon in a large multicenter investigation of neonates with VLBW occurring in only 1.9%. Whereas almost 50% of the study population was characterized for sepsis because of clinical signs, in 98% blood culture reports revealed negative results, but antibiotic treatment was continued fearing false negative results possibly due to maternal antibiotic medication (Stoll et al., 1996). A high rate of failed detection of bacterial growth in blood cultures of VLBW neonates between 27% and 92% could possibly be explained by a transient or intermittent bacteremia as sepsis is known to be a dynamic process (Haque, 2010). Beyond that, interpreting results when organisms are of low or questionable virulence as pathogen or possible contamination- frequently occurring during blood collection (Pourcyrous et al., 1993)- is

*1.1.1. The White blood cell count (WBC) as primary diagnostic tool in neonatal sepsis* 

Although several studies have shown a poor predictive value performing a single WBC as a screening method in asymptomatic neonates with infectious risk factors or later on culture proven sepsis (Ottolini et al., 2003; Rozycki et al., 1987), the assessment of a complete blood cell count (CBC) is usually performed as a routine method to evaluate newborns at risk. The recent introduction of several new parameters to the routine CBC with white blood differential performed by automated hematology analyzer have enabled quantification of cells which were previously solely classified as abnormal flags (Briggs et al., 2003). This refers mainly to the compartment of immature neutrophil granulocytes (IG) which have been detected in various conditions including later stage of pregnancy, steroid therapy, cancer, trauma, or myeloproliferative diseases (Briggs, 2009). They have been considered as helpful early indicators of infectious conditions (Buttarello & Plebani, 2008; Rodwell et al., 1988) and have a long clinical tradition in the diagnosis of bacterial sepsis in neonates

A commonly used index to comprise the fraction of IG in the clinical practice is the IT (immature-to-total-neutrophil)-ratio which is defined as the proportion of the number of immature cells including blasts, band cells, myelocytes, and metamyelocytes to the number of mature neutrophil cells. It is a manual count usually determined by a peripheral blood smear. Already more than three decades ago elevation of IT-ratio was considered to be a useful aid in the diagnosis of neonatal bacterial sepsis. The authors suggested that the

blood culture remains sterile (Guerti et al., 2011).

often difficult.

(Akenzua et al., 1974).

### **1.1. Diagnostic approaches to neonatal sepsis**

Despite a myriad number of scientific studies evaluating the performance of laboratory markers, risk scores, and clinical features in neonatal sepsis, the search for a perfect diagnostic test with high accuracy and reliability still seems to be a quest for the holy grail (Briggs et al., 2000). Still, no single laboratory parameter and none of the newly created clinical risk scores are generally accepted to define the diagnosis of sepsis in its early course with 100% accuracy and confidence (Fowlie & Schmidt, 1998; Rodwell et al., 1988). A systematic review of the literature of 194 studies reporting on different diagnostic tests to predict the presence or absence of bacterial infection in infants up to 90 days of age generally described a poor methodological quality (Fowlie & Schmidt, 1998). Even in rigorous studies the accuracy of the tests showed enormous variation and the diagnostic value was considered as limited in this population (Fowlie & Schmidt, 1998). Although blood culture is considered as the gold standard to confirm the diagnosis of sepsis, this method has its limitations in a neonatal - especially in a preterm – population (Chiesa et al., 2004; Fowlie & Schmidt, 1998). In a study to determine the minimum required blood volume to detect bacteremia Schelonka found that a 0.5 mL blood sample –as commonly obtained in neonatal intensive care units (NICU) - is insufficient to obtain sensitive results when the colony count is less than 4/mL. This is of special interest as it has been shown that low-level bacteremia is common in infants less than two months of age accounting up to 68% (Kellogg et al., 1997; Schelonka et al., 1996). Furthermore false negative results can be obtained due to the presence of antimicrobial agents in the blood because of an early onset of treatment based on empirically decision making representing a regular practice in clinical routine (Fowlie & Schmidt, 1998). Because blood culture bottles require sufficient incubation time, results are delayed and can normally be expected within three days, whereas in infants up to an age of 72 hours blood cultures require a longer incubation time. In a retrospective observational study comprising more than 2900 neonates the median time to positivity of blood cultures was significantly shorter in Gram negative (11.2 hours; 8.5-15.7) compared to Gram positive organisms (23.6 hours; 15.3-4.6). These findings could have important clinical implications to optimize antimicrobial therapy. The authors suggest targeting only for Gram positive germs when the blood culture is still negative after 48 hours and to cease treatment in well-being infants without clinical and laboratory signs of infection after 72 hours when blood culture remains sterile (Guerti et al., 2011).

60 Neonatal Bacterial Infection

effects and help to increase cost effectiveness.

**1.1. Diagnostic approaches to neonatal sepsis** 

Considering these data it is comprehensible that many neonatologists hazard the consequences of possibly unnecessary exposure to antimicrobial agents in neonates suspected for sepsis with unspecific symptoms and uncertain infectious state to avoid fatal outcome caused by a delay in treatment. The above-mentioned declined incidence of EOS was mainly caused by a change in the pathogen distribution showing a decline in group B streptococcus (GBS) sepsis but an increase in Escherichia coli sepsis with a rate of 85% of ampicillin resistance (Fanaroff et al., 2007). As the use of broad-spectrum antimicrobial agents like the combination of ampicillin and an aminoglycoside is considered as the optimal treatment of infants with suspected EOS (Polin, 2012), the progressively increasing burden of antimicrobial resistance would actually require a more targeted drug therapy in the future to confine human and economic costs. The often unspecific early symptoms and the potential rapid deterioration necessitate early identification of patients at risk. This should help to avoid a delay in treatment and could prevent further complications. On the other hand, overtreatment of newborn infants with maternal risk factors and uncertain infectious status suspected for sepsis could also be reduced helping to avoid prescription of unnecessary prophylactic broad-spectrum antibiotic medication, to restrain the development of antimicrobial resistance, exposing the patients to possible severe adverse

Despite a myriad number of scientific studies evaluating the performance of laboratory markers, risk scores, and clinical features in neonatal sepsis, the search for a perfect diagnostic test with high accuracy and reliability still seems to be a quest for the holy grail (Briggs et al., 2000). Still, no single laboratory parameter and none of the newly created clinical risk scores are generally accepted to define the diagnosis of sepsis in its early course with 100% accuracy and confidence (Fowlie & Schmidt, 1998; Rodwell et al., 1988). A systematic review of the literature of 194 studies reporting on different diagnostic tests to predict the presence or absence of bacterial infection in infants up to 90 days of age generally described a poor methodological quality (Fowlie & Schmidt, 1998). Even in rigorous studies the accuracy of the tests showed enormous variation and the diagnostic value was considered as limited in this population (Fowlie & Schmidt, 1998). Although blood culture is considered as the gold standard to confirm the diagnosis of sepsis, this method has its limitations in a neonatal - especially in a preterm – population (Chiesa et al., 2004; Fowlie & Schmidt, 1998). In a study to determine the minimum required blood volume to detect bacteremia Schelonka found that a 0.5 mL blood sample –as commonly obtained in neonatal intensive care units (NICU) - is insufficient to obtain sensitive results when the colony count is less than 4/mL. This is of special interest as it has been shown that low-level bacteremia is common in infants less than two months of age accounting up to 68% (Kellogg et al., 1997; Schelonka et al., 1996). Furthermore false negative results can be obtained due to the presence of antimicrobial agents in the blood because of an early onset of treatment based on empirically decision making representing a regular practice in clinical routine (Fowlie & Schmidt, 1998). Because blood culture bottles require sufficient incubation time, Generally, blood culture-proven EOS has been described as quite uncommon in a large multicenter investigation of neonates with VLBW occurring in only 1.9%. Whereas almost 50% of the study population was characterized for sepsis because of clinical signs, in 98% blood culture reports revealed negative results, but antibiotic treatment was continued fearing false negative results possibly due to maternal antibiotic medication (Stoll et al., 1996). A high rate of failed detection of bacterial growth in blood cultures of VLBW neonates between 27% and 92% could possibly be explained by a transient or intermittent bacteremia as sepsis is known to be a dynamic process (Haque, 2010). Beyond that, interpreting results when organisms are of low or questionable virulence as pathogen or possible contamination- frequently occurring during blood collection (Pourcyrous et al., 1993)- is often difficult.

#### *1.1.1. The White blood cell count (WBC) as primary diagnostic tool in neonatal sepsis*

Although several studies have shown a poor predictive value performing a single WBC as a screening method in asymptomatic neonates with infectious risk factors or later on culture proven sepsis (Ottolini et al., 2003; Rozycki et al., 1987), the assessment of a complete blood cell count (CBC) is usually performed as a routine method to evaluate newborns at risk. The recent introduction of several new parameters to the routine CBC with white blood differential performed by automated hematology analyzer have enabled quantification of cells which were previously solely classified as abnormal flags (Briggs et al., 2003). This refers mainly to the compartment of immature neutrophil granulocytes (IG) which have been detected in various conditions including later stage of pregnancy, steroid therapy, cancer, trauma, or myeloproliferative diseases (Briggs, 2009). They have been considered as helpful early indicators of infectious conditions (Buttarello & Plebani, 2008; Rodwell et al., 1988) and have a long clinical tradition in the diagnosis of bacterial sepsis in neonates (Akenzua et al., 1974).

A commonly used index to comprise the fraction of IG in the clinical practice is the IT (immature-to-total-neutrophil)-ratio which is defined as the proportion of the number of immature cells including blasts, band cells, myelocytes, and metamyelocytes to the number of mature neutrophil cells. It is a manual count usually determined by a peripheral blood smear. Already more than three decades ago elevation of IT-ratio was considered to be a useful aid in the diagnosis of neonatal bacterial sepsis. The authors suggested that the

higher the degree of elevated IT-ratio was, the higher was the risk of bone marrow depletion and death from sepsis (Christensen et al., 1981).

The Role of Immature Granulocyte Count

and Immature Myeloid Information in the Diagnosis of Neonatal Sepsis 63

2004; Schelonka et al., 1995). Contrariwise in performing a standard 100-cell manual differential small numbers of IGs are often underestimated as they can often be overlooked in samples of leukopenic patients. Another study highlighted the wide range of inter- and intraobserver variance in microscopic band cell identification: A smear of a blood sample from a septic patient was prepared, stained and a PowerPoint presentation was made twice of 100 random cells and sent to 157 different hospital laboratories in the Netherlands for a leukocyte differential. In the rst survey neutrophils were differentiated in segmented and band neutrophils whereas in the second survey no discrimination was made between segmented and band neutrophils. Albeit the morphologic characteristics of a band cell are well defined, this study showed an enormous intervariability of enumeration of band cells so that the authors recommended to cease quantitative reporting of counted band cells especially in regard to other diagnostic tools like C-reactive protein (CRP), procalcitonin, and cytokines (van der Meer et al., 2006). Hence, several authors consider the manual count as inappropriate as a reference method for detection of IGs (Fernandes & Hamaguchi, 2007;

**2. Automated detection of immature granulocytes- Clinical applicability**

relation to the lymphocyte count (Bernstein & Rucinski, 2011).

Automated measurement of IGC could represent a reliable and utile method in the prediction of bacterial infection in neonates. In a study evaluating 106 samples from patients with an absolute neutrophil count (ANC) less than 2.0 × 109/L measured with an automated 5-part differential hematology instrument the IGC showed a very good precision and accuracy when compared with a flow cytometric neutrophil count using monoclonal antibodies for cell classification (Amundsen et al., 2012). In another investigation of 200 febrile patients suspected to have infection the performance characteristics of automated IGCs in predicting blood culture results and their clinical utility were assessed. The study population included adults, children, infants, and neonates. Measurements were performed using the Coulter Act Diff 5 counter which can perform a 5-part differential leucocyte count and can also numerate the percentage and absolute number of IG using a technology that combines cytochemistry, focused flow impedance, and light absorbance. The means of IGC and the percentage of IG (IG%) between culture positive and negative groups were statistically significant suggesting that they are potential markers for bacteremia. Among the 51 culture positive cases, 49 had an IT-ratio > 0.65% giving a sensitivity of 96.1%. IGC of 0.03×103/µL and IG% of 0.5% offered a sensitivity of 86.3% and 92.2%, respectively. Higher values of IGC > 0.3 and IG% > 3 had a specificity greater than 90%, although the values were infrequent. Receiver operating characteristic (ROC) curves showed that IGC was a better predictor of infection than WBC and ANC in adults and the ratios IGC/WBC and IGC/ANC did not improve the prediction outcome (Senthilnayagam et al., 2012). Another study reported an in parallel increase of IG values to an increase of the ANC and an inverse

In an adult study population including patients suspected for sepsis higher percentages of IGs have been observed in infected than in non-infected patients and in patients with positive than patients with negative blood cultures (Ansari-Lari et al., 2003). Also in preterm

Senthilnayagam et al., 2012).

In a retrospective multicenter cohort analysis including 166092 neonates with suspected EOS admitted to 293 NICUs in the United States low WBC counts, low absolute neutrophil counts, and high IT-ratios were associated with increasing odds of infection. Elevated ITratios of >0.2, >0.25, and >0.5 had low sensitivities (54.6%, 47.9%, 21.9%, respectively), but were associated with relatively high specificities (73.7%, 81.7%, 95.7%, respectively) and negative predictive values (NPV) (99.2%, 99.2%, 99.0%, respectively), whereas positive predictive values (PPV) were low (2.5%, 3.2%, 6.0%, respectively). The authors concluded that due to the low sensitivity these CBC-derived indices do not represent reliable diagnostic markers to rule out EOS in neonates (Hornik et al., 2012). The very high negative predictive accuracy of more than 90% is in contrast to high rates of elevated IT-ratios between 25% and 50% in non-infected infants (Polin, 2012).

In a large historical cohort study comprising more than 3100 neonates, patients were evaluated for EOS. In this study a normal WBC was defined as an IT-ratio of less than 0.2 and a total WBC between 6000 and 30000/µL. Two serial normal WBCs with normal ITratios performed 8 to 12 hours apart and a negative blood culture at 24 hours were predictive of healthy newborns in the evaluation of EOS in the rst 24 hours after birth and showed a negative predictive value of 100%. The sensitivity of 2 normal WBCs and a negative blood culture at 24 hours was 100%, as was NPV. The specificity was 51%, and the PPV was 8.8% (Murphy & Weiner, 2012). These results suggest that combinations of parameters and repeated performance of diagnostic tests are likely to increase accuracy.

In a review article Cornbleet reported a wide range of sensitivity and specificity for the ITratio and predicted a possible replacement by the measurement of newly created markers for infection such as inflammatory factors, adhesion molecules, cytokines, neutrophil surface antigens, and bacterial DNA (Cornbleet, 2002). Recent advances in basic science of predicting and diagnosing neonatal sepsis are developing towards more and more sophisticated approaches like the determination of proteomic inflammatory biomarkers in amniotic fluid (Buhimschi et al., 2009). Regarding these new techniques, the diagnostic value of traditional laboratory methods has to be critically analysed. However, comparing these new methods for the detection of neonatal sepsis with the measurement of WBCs including the assessment of the IG count (IGC) as well as the IT-ratio, the additional sample volumes, delayed availability of results, and considerably higher labour and laboratory costs should be taken into account.
