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Invited Review

Effective Biomarkers for Diagnosis


of Neonatal Sepsis
Vineet Bhandari
Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut

Corresponding Author: Vineet Bhandari, MD, DM, Yale University School of Medicine, Yale Child Health Research Center,
Room Number 219, PO Box 208081, 464 Congress Ave, New Haven, CT 06520. E-mail: vineet.bhandari@yale.edu.
Received January 21, 2014; accepted May 22, 2014; electronically published June 24, 2014.

Infection in neonates continues to be a global problem with signicant morbidity and mortality. The diagnosis of
neonatal sepsis is complicated by nonspecic clinical symptomatology, a high-false negative rate, and a delay in
obtaining blood culture results. An ideal biomarker needs to have a high degree of accuracy in recognizing the
presence or absence of denite infection at an early stage, to guide the initiation and duration of antibiotic
therapy. The diagnostic utility of the following biomarkers seems to be most practical in the early (interleukin
[IL]-6, IL-8, tumor necrosis factor-alpha, neutrophil CD64), mid ( procalcitonin) and late (C-reactive protein)
phases of neonatal sepsis. Future research studies to assess reliability of these biomarkers should be (1) adequately
powered for sample size and (2) use the gold-standard denition of blood-culture proven pathogen-specic
sepsis. Signicant advances in diagnostic accuracy of novel biomarkers to allow early, accurate, and cost-effective
identication of pathogens responsible for neonatal sepsis is anticipated in the next 5 years.
Key words. CRP; cytokines; infection; neutrophil CD64; newborn; procalcitonin.

Death due to infections remains a major contributor to of resistant microorganisms and mortality in neonates with
mortality in children younger than 5 years of age world- denite sepsis.
wide [1]. The incidence and etiology of early- and Multiple factors can inuence the concentration of a spe-
late-onset sepsis in neonates is variable across countries cic biomarker, resulting in variations in the diagnostic ac-
[27], which necessitates that antibiotic therapy be tailored curacy of the same, when compared across studies [16].
in an institution-specic manner. However, the difculties Such factors can range from differences in the study popu-
in conrming the diagnosis of neonatal sepsis have led to lation (antenatal/perinatal conditions, gestational age
the use of a variety of antibiotics for variable durations [GA], birth weight [BW], etc), mode and timing of data col-
leading to the emergence of antibiotic-resistant microor- lection, the nature of the infectious agent causing the in-
ganisms [812]. Understandably then, there has been sig- ammatory response in the infant, which, in turn,
nicant interest in identication of specic biomarkers of inuences the severity of the presentation and progress of
neonatal sepsis [1315]. In the current context, a bio- the disease, to the denitions used for healthy controls
marker is dened as any measurable parameter that pro- as well as for conrmed/presumed/suspected sepsis. In mul-
vides meaningful information about the diagnosis of ticenter studies, comparability of reliability of any single
neonatal sepsis. An ideal biomarker for neonatal sepsis test as a biomarker of neonatal sepsis among institutions
would not only have a high degree of accuracy in recogniz- may be diminished by variation in disease severity.
ing the presence (or absence) of denite infection at an Early-onset sepsis (EOS) is usually due to transplacental,
early stage, but it would also be useful to guide the duration ascending, or intrapartum transmission in the perinatal pe-
of antibiotic therapy. Given the risk of delaying antibiotics riod shortly before or during birth, up to postnatal (PN) day
in neonates with denite sepsis, such a biomarker should 3 [17, 18]. Late-onset sepsis (LOS) is acquired by horizontal
be able to make the determination in a reasonably short pe- transmission in the home, hospital, or in the community
riod (within a few hours). Thus, an ideal biomarker would after PN day 4 [17, 18]. By convention, these cutoff values
determine the initiation and length of exposure of antibiot- have been used and are reected in the studies summarized
ics in a safe manner, decreasing the potential of emergence below. Given the problems associated with smaller blood

Journal of the Pediatric Infectious Diseases Society, Vol. 3, No. 3, pp. 23445, 2014. DOI:10.1093/jpids/piu063
The Author 2014. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society.
All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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Biomarkers in Neonatal Sepsis 235

volumes taken from neonates and exposure to antenatal an- preterm, access to the amniotic uid (AF) provides an op-
tibiotics (to mention 2 reasons), the chances of getting a pos- portunity to search for biomarkers [19]. Although AF col-
itive blood culture (considered the gold-standard for lection is not routinely practiced for detection of EOS in
diagnosis of sepsis) are markedly reduced. There is no stan- some centers and is associated with the inherent risks
dard denition of clinical sepsis in neonates (which can vary (for example, bleeding) of being an invasive procedure, it
from relying on limited clinical symptomatology to includ- does allow unique access to the intrauterine environment.
ing selective laboratory and radiological investigations), Although earlier studies assessed specic cytokines [20,
and this inconsistency adds another confounding variable 21], recent investigations into the application of proteomic
when assessing biomarker studies in neonatal sepsis. analyses of the AF have shown signicant promise [22].
The search strategy utilized PubMed, focusing on pub- The Mass Restricted (MR) score was generated from the
lished studies restricted to those in English and after AF using a single surface-enhanced laser desorption ioniza-
2008, using the search words newborn or neonatal, tion time-of-ight mass spectrometer instrument [22].
and sepsis, and biomarkers. Further selection was Surface-enhanced laser desorption ionization time-of-ight
done to restrict to the most promising and novel biomark- outputs are sequences of values with the molecular weight
ers. Earlier (before 2008) original and review studies were on the horizontal axis and normalized peak intensity on
accessed and referred to wherever appropriate. The present the vertical axis; the MR score was devised using a stepwise
review will be mostly limited to studies related to identi- strategy based on lter preferences applied in a sequential
cation and testing of the clinical utility of select biomarkers manner to result in a numeric score [19]. A priori a categor-
of neonatal sepsis in the last 5 years, primarily in the neo- ical value of 1 was assigned if a particular peak was present
natal intensive care unit (NICU) population. The focus will and 0 if absent. Proteomic mapping of the AF revealed a
be on biomarkers with reporting of details noted above, as characteristic ngerprint generating a MR score utilizing
available. Most cytokine levels are not expected to be nor- the presence of 4 biomarkers: neutrophil defensin-1 and
mally distributed in studies done in the neonatal popula- defensin-2, along with calgranulins A (S100A8) and C
tion. Although in some studies the actual cytokine levels (S100A12). The study specied that a score of 34 indicat-
have been used to run the analyses, in others receiver oper- ed the presence of inammation, whereas a score of 02
ator characteristic (ROC) curves were used to generate the excluded it. Thus, the population was stratied based on
cutoff values. The latter approach will be used in this man- the severity of inammation (MR = 0 indicated no in-
uscript, unless otherwise explicitly stated. The primary ammation; MR = 12 indicated minimal inammation;
objective of the present review is to summarize the informa- and MR = 34 indicated severe inammation) [22].
tion about biomarkers that have been reported in real- Women with MR scores of 34 were more likely to deliver
life NICU settings, in order for a list of selected biomark- neonates with EOS [2224]. These studies were conducted
ers be generated, which would be the focus of further test- with a prospective collection of consecutive samples, with
ing in a prospective manner with adequately powered the neonates being admitted to and monitored in the
sample sizes using the gold-standard test of positive NICU. Early-onset sepsis was dened as being conrmed
blood culture as the reference point. The majority of the (culture positive) or suspected (based on presence of vali-
studies reported in this review have been prospectively dated hematologic criteria when 2 or more of the following
done in the NICU setting. Although methodological differ- were observed: absolute neutrophil count [ANC] <7500/
ences do exist among studies (for example, in the diagnosis mL or >14 500/mL, absolute band count [ABC] >1500/
of presumed or clinical sepsis) and could account for some mL, immature/total neutrophil ratio [I:T ratio] >0.16,
of the variability of the results, if a biomarker is going to be platelet count <150 000 cells/mm3, or an abnormal spinal
potentially clinically useful in the NICU setting, there should tap, if done) [2224]. Neonatal hematological indices and
be enough of a signal from multiple studies for it to stand EOS signicantly correlated with the MR score, even after
out. This method was a practical approach that I believe adjusting for GA [22]. Although clinicians are likely to
would narrow down the eld to selective biomarkers for in- treat a symptomatic infant, given a history of clinical cho-
vestigators to focus their energies upon in the next 5 years. rioamnionitis, if data on AF biomarkers (as noted above)
could be made available with a short turn-around time
(TAT), it would be an important step towards a more ratio-
Early-Onset Sepsis: Amniotic Fluid
nal use of antibiotics.
Given the well known association of presence of infection It is well known that using traditional culture methods
and inammation in the intrauterine environment predis- underestimates the infectious etiology of intra-amniotic
posing to EOS to neonatal sepsis, especially those born inammation (IAI). Towards that end, metagenomic

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236 Bhandari

techniques and sequencing technology for specic identi- under the curve (AUC) ROC curves (IL-6, 0.860.9;
cation of species of microorganisms that do not grow in IL-8, 0.790.87). The last was a prospective case-control
culture, by sequencing 16S ribosomal RNA (rRNA), is study of 120 consecutive preterm infants in a NICU setting;
now possible [25]. Samples were obtained from a cohort EOS was dened as positive cultures (n = 20) and highly
of consecutive patients enrolled at Yale-New Haven probable sepsis (n = 20), using clinical, laboratory criteria
Hospital who had paired AF and cord blood available (including hematological and C-reactive protein [CRP] val-
for analysis (n = 161). To avoid selection bias, cases fulll- ues) [29]. Cord blood levels of 25100 pg/mL for IL-6 and
ing the clinical group requirements were selected consecu- 50300 pg/mL for IL-8 were the range of cutoffs used in
tively based on the availability of at least 1 mL of umbilical the studies reviewed [26, 27]. It is also important to remem-
vein cord blood for DNA extraction and other research ber that IL-6 levels can be modied by illness severity, in-
analyses. The study groups were designed using the results cluding perinatal asphyxia, in noninfected infants [30, 31].
of AF and neonatal blood bacterial cultures as reported by Interleukin-8 levels in cord blood can vary by GA [32]. In
the microbiology laboratory. The following groups were addition to IL-6 and IL-8, procalcitonin (PCT) was found
studied: Group 1: premature newborns with neonatal to be superior to CRP and tumor necrosis factor-alpha
blood culture conrmed early-onset neonatal sepsis (me- (TNF-) as diagnostic biomarkers for EOS [27]. The
dian GA at delivery, 25 weeks; n = 6); Group 2: premature derived cutoff values of PCT used in the cord blood studies
newborns with negative neonatal blood culture but pre- ranged from 0.5 to 1.22 ng/mL (AUC, 0.80.96) [27].
sumed EOS and positive IAI (27 weeks; n = 16); Group More importantly, IL-6, IL-8, and PCT levels could be
3: premature newborns with negative neonatal blood cul- measured in 50100 L plasma with TAT of 90 minutes
ture but presumed EOS and negative IAI (32 weeks; n = 7); or less [27].
Group 4: premature newborns without EOS and no IAI
(32 weeks; n = 7); Group 5: term healthy newborns (39
Early-Onset Sepsis: Peripheral Blood
weeks; n = 8) delivered by elective Cesarean within the
same time period. This last group served as a technical con- It would be important to mention that whereas cord blood
trol for handling and analysis of the samples. Six, 15, 5, samples are reective of the intrauterine environment lead-
and 1 patient had histological chorioamnionitis in ing to EOS, biomarkers measured in peripheral blood col-
Groups 1 to 4, respectively, with all 21 samples in lected at variable time points in the rst 3 days of PN life
Groups 1 and 2 only having positive results with the could be potentially inuenced by a variety of factors.
culture-independent method from AF or cord blood However, administration of antenatal antibiotics would
samples. Five (of 6 in Group 1 only) neonatal blood cul- impact on the incidence of detecting positive blood cultures
tures correlated with positive results with the culture- irrespective of the source of the blood sampling. With the
independent method from AF or cord blood samples. It high rate of negative blood cultures usually noted in the
was noted that 72% of the microbial species identied classic scenario of EOS, it is imperative that testing of bio-
(Escherichia coli and Fusobacterium nucleatum being the markers be done against the benchmark of the positive
most common) in the cord blood matched those in the blood culture, before being put into clinical practice.
AF. Using the16S rRNA sequencing approach, the paired Traditionally, clinicians have relied on using the com-
samples were 99.9%100% identical [25]. Thus, the plete blood count (CBC), despite studies reporting on the
strength of this technique to recognize unique microorgan- unreliability of hematologic indices (namely, total white
isms that are otherwise difcult to detect using convention- blood cell count [WBC], ANC, ABC, I:T ratio, and platelet
al culture approaches provides us with the potential for an count) for diagnosis of EOS [33]. However, in a retrospec-
early diagnosis of EOS [25]. tive cross-sectional study (n = 67, 623) evaluating the CBC,
although there were lower mean values in some of the com-
ponents of the CBC [WBC and ANC], immature neutro-
Early-Onset Sepsis: Cord Blood
phils were higher in neonates with infection, with no
A variety of cytokines and other acute-phase reactants have difference in platelet counts [34]. The ability of WBC and
been investigated for their potential for biomarkers in um- ANCs to differentiate the cases of culture-proven EOS did
bilical cord blood for diagnosis of EOS [26, 27]. As with get better with increasing PN age, but these indices were
multiple previous studies [26], more recent reports most useful when their values were very low [34]. Based
[24, 28, 29] have also suggested that cord blood interleukin on the above noted ndings, the study investigators con-
(IL)-6 and IL-8 seem to have the best discriminatory ability cluded that the CBC indices were most helpful in detecting
to diagnose EOS using cutoff values derived from area EOS after 4 hours; hence, if possible, waiting to initiate

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Biomarkers in Neonatal Sepsis 237

antibiotic therapy after that PN age would be preferred if in a prospective NICU-based study enrolling consecutive
relying on the CBC results. If there should be signicant infants (n = 580 evaluated for EOS) [43]. This test can be
concern for EOS before that age, antimicrobial therapy done with a minimal amount of blood (50 L) with a
should be begun immediately, after sending off the CBC rapid TAT (2 hours) in clinical hematological laborato-
and blood cultures [34]. ries having dedicated access to ow cytometry [43].
Earlier studies have suggested that IL-6 derived cutoff Novel biomarkers with potential usefulness for diagnosis
levels of 2050 pg/mL in the peripheral blood of neonates of neonatal EOS have been shown in Table 1.
seem to be the most promising marker for neonatal EOS
[26], although serial measurements may be necessary for
Late-Onset Sepsis: Peripheral Blood
conrmation of infected neonates ( prospectively collected
samples in a NICU setting with infection diagnosed based Use of the CBC in 1 large retrospective study, which ob-
on clinical, laboratory, and culture criteria) [35]. A recent tained administrative hematological data collected in a pro-
prospective study (n = 123; 29 with conrmed/suspected spective manner from 293 NICUs, essentially conrmed
sepsis, 94 with no sepsis) has suggested that using a combi- that high and low WBC counts, high ANCs, high I:T neu-
nation of derived cutoff levels of IL-6 ( >250 pg/mL) and trophil ratios, and low platelet counts were associated with
PCT ( >25 ng/mL) was a reasonable approach to diagnose LOS [44]. Associations were weaker with increasing PN
EOS (culture-proven or strongly suspected) [36]. age at the time of the culture. Sensitivities were low for
In a recent detailed review of the use of CRP as a bio- all index cutoffs analyzed, whereas specicity was general-
marker in EOS, it was reiterated that physiologic dynamics ly high. The authors concluded that no CBC count index
(including GA) as well as maternal and perinatal factors possessed adequate sensitivity to reliably rule out
can inuence the levels of CRP in the rst 3 days of PN culture-proven LOS in neonates [44].
life [3740]. C-reactive protein has the best diagnostic ac- Most investigators in previous studies have evaluated
curacy (derived cutoff value of 10 mg/L) when combined IL-6 levels in neonates as a marker for LOS [26]. Using
with other biomarkers (for example, but not limited to the same cutoff value (25 pg/mL) as for cord blood values
PCT, IL-6, and IL-8) [37]. Interleukin-6 and IL-8 levels for EOS seems to have promise in the diagnosis of LOS;
can vary by GA and PN age [39, 41]. Serial determination however, although the measurements have good sensitivi-
of CRP has been reported to have 99% negative predictive ties, the test is not that specic [26]. The clinical usefulness
value (NPV) in identifying noninfected neonates, although of IL-6 is reduced by its very short half-life, resulting in a
this should not replace clinical judgment and blood culture rapid decrease in sensitivity [15]. The same disadvantage
results [37]. The magnitude of the CRP response was noted occurs with IL-8 [45]. Interleukin-6 and IL-8 levels can
to be higher in Gram-negative versus Gram-positive infec- vary by GA and PN age [39, 41].
tions; among the latter, signicantly lower median values Interleukin-6 or TNF- (both had good sensitivity) com-
were reported for coagulase-negative staphylococci [37]. bined with CRP (which had good specicity) was a good
Neutrophil CD64 (nCD64), a high-afnity Fc receptor diagnostic marker for LOS (culture-proven and/or clinical
that increases when neutrophils are exposed to infectious criteria) in a prospective study of preterm neonates [46]. In
stimuli ( primarily secondary to bacterial and fungal an independent study median, CRP levels and CBC were
agents), has been studied as a marker for EOS. Most stud- useful for the diagnosis of conrmed or probable LOS
ies have reported nCD64 to have good diagnostic accuracy and comparable with median IL-6 and TNF- concentra-
[42, 43]. For blood-culture proven EOS, the CD64 index tions [47].
with a derived cut-point value of 2.38 had sensitivity, spe- As with EOS, most studies have reported nCD64 to be
cicity, and NPV of 100%, 68%, and 100%, respectively, an accurate diagnostic marker for LOS [43, 48]. For

Table 1. Promising Novel Biomarkers of EOS in Neonates


Biomarker Source Sample Size* Cutoff Value / AUC Sensitivity (%) Specificity (%) PPV (%) NPV (%)
MR Score [23] AF 22 of 97 Presence of 1 of 4 components 5586 2880 2644 8688
TBARS [29] Cord blood 40 of 120 0.88
SAA [101] Peripheral blood 23 of 104 8 mg/L / 0.987 96 95 85 99
nCD64 [43, 81] Peripheral blood 207 of 623 1.63%2.38% / 0.720.89 67100 6768 250 80100
Abbreviations: AF, amniotic fluid; AUC, area under the receiver operating characteristic curve; EOS, early-onset sepsis; MR, mass restricted; nCD64, neutrophil CD64;
NPV, negative predictive value; PPV, positive predictive value; SAA, serum amyloid A; TBARS, thiobarbituric acid reactive substances.
*Confirmed or suspected sepsis/total number of infants.

Calgranulin A, C, Defensin 1, 2.

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238 Bhandari

culture-proven LOS, the CD64 index with a derived cut- neonatal sepsis [55, 56, 59]. Polymerase chain reaction
point value of 3.62 had sensitivity, specicity and NPV (PCR) allows detection of bacterial DNA [55, 56, 59].
of 75%, 77%, and 96%, respectively, in a prospective Polymerase chain reaction is a technique to amplify a single
NICU-based study enrolling consecutive infants (n = 417, or a few copies of a DNA strand across multiple orders of
evaluated for LOS) [43]. In a recent study, with a derived magnitude, generating thousands to millions of copies of a
cutoff of 2.85, the sensitivity was 87.5% and specicity of particular DNA sequence. Approaches to enhance the abil-
100% for nCD64 index in blood-culture proven LOS [49]. ity to detect neonatal sepsis include [56]: (1) bacterial DNA
Among noninvasive approaches, using a heart rate char- amplication from whole blood. In this approach, whole
acteristics index was shown to be useful for detecting LOS blood is utilized to screen for a highly conserved 16S ribo-
[50]. In a randomized clinical trial utilizing this approach, somal DNA (rDNA) target that is present almost universal-
there was a reduction in mortality from 10.2% to 8.1% ly in all bacteria but not in human cells. This technique has
(P = .04) in infants who had the heart rate characteristics the advantage of rapid diagnosis but suboptimal sensitivity
index visible to clinicians [51]. However, this approach and specicity. (2) Pre-enrichment of whole blood before
led to 10% more blood cultures being sent and 5% more target amplication is the next approach. Because increas-
days on antibiotics in the group with the heart rate charac- ing the volume of the collected blood sample is not an at-
teristics index display [51]. tractive option in the NICU population, using a tryptic
An under-utilized, noninvasive approach is to monitor soya broth (TSB) incubation enrichment protocol in con-
core-peripheral temperature differences in neonates at risk junction with PCR and pyrosequencing was utilized. In
for sepsis. This has been shown to be a fairly accurate pre- this process, whole blood is incubated with TSB for up to
dictor of LOS (culture proven or necropsy ndings) in devel- 5 hours before nucleic acid extraction and then screened
oped [52] as well as in resource-limited countries [53]. There for bacterial 16S rDNA after the preamplication step.
was signicant widening of the rectal-sole and axillary-sole Pyrosequencing determines the order of nucleotides (se-
temperatures in the preterm neonates with sepsis quence) in the DNA by detecting the light emitted upon in-
(culture-proven or based on clinical or laboratory criteria) corporation of the next complementary nucleotide based
(P < .001) [53]. With an overall accuracy of 90.9%, a rectal- on the fact that only 1 of 4 (A, T, C, or G) possible nucle-
sole temperature difference of 2.3C (100% sensitivity) or otides are added and available at any one given time so that
3.2C (100% specicity) was a useful marker to differen- only 1 can be incorporated on the single-stranded template.
tiate normothermic preterm neonates with or without sep- The percentage of near-term infants with culture-negative
sis. Using the axillary-sole temperature difference, the results was 98.6% (1216 of 1233), whereas those with
respective values were 2.2C and 3.0C [53]. These re- PCR-negative results was 97% (1196 of 1233). Compared
sults have been recently replicated in a prospective observa- with blood culture, PCR demonstrated a high NPV (99.2%)
tional study with proven or probable LOS [54]. and specicity (97.5%). However, the 16S rDNA PCR
assay failed to detect 10 of the 17 cases of culture-proven
Early- and Late-onset Sepsis: Peripheral Blood sepsis. (3) Another approach is PCR-based pathogen iden-
Molecular Assays. Using mass spectrophotometric techniques tication from positive blood culture bottles. In this ap-
to identify bacteria have the possibility of earlier iden- proach, bacterial DNA is isolated from the specimen,
tication (within 1 hour) of pathogens but modest amplied by PCR, detected, and the identity of the patho-
sensitivities (66%80%), poor yield when there is low gen conrmed by hybridization and an alkaline phospha-
bacterial density, and risk of misidentication of path- tase reaction on a membrane strip. Others have used a
ogens, especially in situations of polymicrobial growth multiplex-PCR-enzyme-linked immunosorbent assay or
are signicant limitations [55, 56]. The commonly used combined real-time PCR with pyrosequencing. Although
approach is that of matrix-assisted laser desorption studies reported variable sensitivity for pathogen detection
ionization time-of-ight mass spectrophotometry (MALDI- (60%100%) highlighting the challenges of optimizing
TOF MS). In this process, ionization of a specic sample DNA extraction from small sample volumes, 1 study did
(in this case, the pathogen that can be detected in a positive show a net gain of 10.7 hours in diagnosing sepsis. (4)
blood culture) results in a specic mass spectrum plot The nucleic acid sequence-based amplication (NASBA)
(intensity vs mass-to-charge ratio) that determines its iden- approach had sensitivities and specicities ranging from
tity. Additional details of the sample preparation and the 96% to 100% for bacterial and fungal (Candida) isolates.
MALDI-TOF MS technique have been published [57, 58]. In the NASBA process, an isothermal nucleic acid ampli-
Molecular techniques to detect the presence of bacterial cation assay that preferentially amplies RNA targets al-
DNA have been utilized to enhance the diagnostic yield in lows rRNA to be expressed at much higher number of

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Biomarkers in Neonatal Sepsis 239

copies compared with PCR. (5) Nonamplication-based at 68 hours, and remaining elevated for at least 24 hours
uorescence in situ hybridization (FISH) studies have re- [15, 45]. In neonatal bacterial sepsis, plasma PCT con-
vealed sensitivities and specicities ranging from 99% to centrations can increase up to 1000 ng/mL, and has
100% in 2 studies [56]. The FISH approach uses probes been shown to have signicant correlations with illness
that have been developed against specic regions (DNA, severity as well as survival, but decreases to normal
RNA) of the targeted pathogen of interest. A meta-analysis values by day 3 [66, 68]. In addition, compared with
of 23 studies utilizing molecular assays for detecting neo- CRP, the increase in PCT levels is much faster. Hence,
natal sepsis has been published [60]. Mean sensitivity and PCT would be preferred over CRP, as a diagnostic
specicity were 90% and 96%, respectively. Real-time biomarker for newborn sepsis [66]. However, the disad-
PCR and broad-range conventional PCR had higher sensi- vantages of PCT measurements include the fact that refer-
tivity and specicity than other assays. Limitations include ence ranges and thus cutoff values are impacted upon by
need for collection of samples by sterile venipuncture, po- GA, PN age, and additional clinical conditions [15].
tential for contamination during collection and processing, Elevation of PCT levels has been noted up to 48 hours
need effective lysis of bacterial cells, and competition with after normal birth. A variety of common neonatal disor-
human DNA if WBC counts are high [61]. The authors ders of a noninfectious etiology (ie, perinatal asphyxia,
concluded that molecular assays do not have sufcient sen- respiratory distress syndrome, and pneumothorax) have
sitivity to replace microbial cultures in the diagnosis of neo- PCT concentrations less than [30] or similar [66] to that
natal sepsis but may perform well as adjunctive tests [60]. of neonatal sepsis. Furthermore, ante-, peri-, and postna-
Cytokines and Acute-Phase Reactants. Investigators have noted tal administration of antibiotics can impact on PCT
that TNF- was the best diagnostic test for sepsis in the levels [66].
NICU (11 with positive cultures, 4 clinical criteria) Investigators evaluated PCT, IL-10, and nCD64 as diag-
[62], whereas others evaluating IL-6 on the 1st day of nostic markers of EOS and LOS (dened as culture-proven
symptoms with CRP-positive (measured after 4872 or with clinical signs and CRP >10 mg/L with antibiotic
hours) sepsis cases noted a sensitivity of 77%, specicity therapy for >10 days) and found that the best combination
of 74%, PPV of 80%, and NPV of 70% [63]. Fungal was of IL-10 (derived cutoff value of >17.3 pg/mL) and
infections in neonates resulted in signicantly higher IL-6 nCD64 (cutoff derived value of >2.6%) [69]. The use of
and CRP levels, compared with those with bacterial IL-6 (derived cutoff value of >21.5 pg/mL) and CRP (de-
sepsis [64]. In a NICU study evaluating CRP, IL-6 and rived cutoff value of >5.82 mg/L) gave sensitivities of
immunoglobulin (Ig)M as diagnostic markers for 75% and 71%, specicities of 82% and 97%, PPV of
neonatal sepsis (culture-proven or suspected based on 92% and 99% and NPV of 52% and 49%, respectively,
clinical and laboratory criteria), it was reported that a for blood-culture proven sepsis [70].
CRP of >4 mg/dL gave the best derived cutoff value with In a meta-analysis of 22 studies that evaluated the PCT
95.7% sensitivity, 88.9% specicity, 78.6% PPV, and test for the diagnosis of EOS at different time points (birth,
98% NPV [65]. The sensitivity of IgM as a single test at 012 hours, 1224 hours, and 2448 hours) and LOS all
a cutoff derived value of 10 mg/dL was 91.3%, with a showed moderate accuracy (Q* = 0.79, 0.86, 0.81, 0.82,
low specicity of 45% [65]. Likewise, the IL-6-derived and 0.77, respectively) [71]. The PCT test was more accu-
cutoff value of 18.2 pg/mL was associated with sensitivity rate than the CRP test for the diagnosis of EOS and LOS
and specicity of 87% and 50%, respectively [65]. [7173]. In a systematic review and meta-analysis of 16
C-reactive protein continues to be used as a biomarker studies (involving 1959 neonates) that utilized PCT
for sepsis in multiple studies. It is a late biomarker with (using derived cutoffs ranging from 0.5 to 5.75 ng/mL)
a high specicity but poor sensitivity [15, 45]. All studies as a biomarker for diagnosis of culture-proven or clinical
on severe, invasive bacterial infections in children report sepsis, the authors concluded that PCT had a higher diag-
higher sensitivities and specicities of PCT than for CRP nostic accuracy for LOS than EOS, with the AUC being
[66]. C-reactive protein decreases to its normal values 0.95 and 0.78, respectively, with an overall AUC of
after 37 days [66]. C-reactive protein levels have been 0.87 [74]. In a recent review, the derived cutoff values of
reported to be inuenced by GA and noninfectious PCT for diagnosis of EOS and LOS ranged from 0.5 to
conditions [67]. >98 ng/mL [68]. Hence, caution must be urged against
Procalcitonin is another extensively studied acute- making rm conclusions given the heterogeneity of the
phase reactant with the advantages of quickly increasing studies included and the lack of uniform denition of
within 4 hours of exposure to bacterial products, peaking sepsis [68, 74].

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240 Bhandari

By activating the complement system, mannose-binding sensitivity of 95% and a specicity of 83%, with a NPV of
lectin (MBL) results in opsonization of the pathogen; 86% [69]. For further enhancing the sensitivity to 100%
hence, decreased MBL would hamper clearance of micro- for the diagnosis of EOS as well as LOS, it would appear
organisms by the immune system [75]. Among 8 prospec- that the combination of nCD64 with specic cytokines
tive studies evaluated, although 1 study had decreased (IL-6 or IL-8) and CRP are presently the most promising
median MBL levels (170 vs 1450 g/L), 2 independent candidates [81].
studies reported MBL concentrations of 400700 g/L, In a recent study, use of nCD64 (utilizing a derived cut-
in neonates with proven sepsis. The authors concluded off value of 5655 antibody-phycoerythrin molecules
that neonates with low MBL levels were associated with bound/cell measurement) for daily surveillance detected
culture-conrmed sepsis [75]. LOS/necrotizing enterocolitis 1.5 days before clinical pre-
In studies that have evaluated both EOS and LOS, sentation, but at the expense of performing 41% additional
nCD64 has been reported to have high sensitivities sepsis evaluations [82]. Some of the promising novel bio-
and NPV [43, 7681]. Sensitivity NPV of IL-6, CRP, markers for LOS in neonates have been summarized in
and nCD64 were 80.0%90.6%, 80.0%88.8%, and Table 2.
88.6%94.0%, respectively, for diagnosis of sepsis
(culture-proven or using clinical/validated hematological
Antibiotic Stewardship
criteria) in a prospective observational NICU study [79].
Combining all 3 tests increased the sensitivity to 100%; Prompt diagnosis of sepsis and initiation of antibiotic (and
however, specicity and PPV decreased to 62.1% and appropriate supportive) therapy are critical in the neonatal
55.5%, respectively [79]. Using the database of the largest population because delays can worsen outcomes [83]. It is
study done to date on this biomarker in neonatal sepsis routine practice in the NICU to start broad-spectrum anti-
[43], a 2.19% nCD64-derived cutoff value for clinical biotics, after a sepsis work-up, while awaiting results of the
LOS (dened using hematological indices) had sensitivity, blood cultures sent [8385]. Antibiotic use, whether mea-
specicity, and NPV values of 78%, 59%, and 81%, re- sured in courses or days of antibiotic treatment, was 9- to
spectively [81]. The study also reported on derived cutoff 14-fold higher in neonates worked up for sepsis compared
values based on BW. For those with a normal BW, it was with those treated for central-line-associated blood stream
2.05, whereas for infants with low BW (LBW; <2500 g) infections [86]. This increased exposure of antibiotics to
and very LBW (<1500 g), the cutoff values were 2.34 and neonates in the NICU is not without consequences.
3.13, respectively [81]. It is likely that differences in the cut- Besides separating infants from the parents (while admitted
off values for nCD64 in EOS and LOS are reective of the to the NICU), there is also the potential risk for develop-
differences in the predominant microorganisms in each ment of antibiotic resistance and changes in the micro-
scenario, with the BW categories as a surrogate marker biome of these neonates [83, 87, 88]. In fact, prolonged
for the maturational aspects of the immune response. antibiotic exposure has been associated with the develop-
For blood culture-proven sepsis, among the hematolog- ment of antimicrobial resistance [87], necrotizing enteroco-
ical indices, the combination of either the ABC or ANC litis [8890], LOS [90], prolonged hospitalization with its
with nCD64 had the highest sensitivity (91%) and specif- attendant expenses [91], and increased mortality [90].
icity (93%) [81]. Combining nCD64 with PCT provided a Although it has been suggested that serial measurements

Table 2. Promising Novel Biomarkers of LOS in Neonates


Biomarker Sample Size* Cutoff Value / AUC Sensitivity (%) Specificity (%) PPV (%) NPV (%)
nCD64 [43, 81] 204 of 533 2.19%3.62% / 0.730.83 7578 5977 2954 8196
SAA [102] 123 of 163 > 6.8 mg/dL / 0.710.88 44.776.4 100 100 38.858
ApoSAA [103] 42 of 73 0.199 100 61 75 100
Hepcidin [104] 17 of 44 > 92.2 ng/mL 76 100 100 87
Calprotectin [105] 62 of 201 1.7 g/mL 89 96 98 80
IaIp [106] 45 of 573  177 mg/L / 0.94 89.5 99 95 98
CD11b [107] 65 of 77 290 fluorescent units 75 100 100 86
Abbreviations: AUC, area under the receiver operating characteristic curve; CD11b, cell surface marker on neutrophils; EOS, early-onset sepsis; IaIp, interalpha
inhibitory proteins; LOS, late-onset sepsis; nCD64, neutrophil CD64; NPV, negative predictive value; PPV, positive predictive value; SAA, serum amyloid A.
*Confirmed or suspected sepsis/total number of infants.

Includes 10 infants with EOS.

Composite score using proapolipoprotein CII and the des-arginine variant of SAA.

LOS and necrotizing enterocolitis used as a composite outcome.

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Biomarkers in Neonatal Sepsis 241

of CRP may dictate the duration of antimicrobial ther- could be extremely variable. The timings suggested are
apy in the NICU [15, 45], it has not yet been proven to based on experimental animal data and sequential mea-
do so [66]. surements conducted in closely monitored settings (ie,
Among the most promising biomarkers of neonatal sep- NICUs). Promising early phase (212 hours) biomarkers
sis that have strong potential to reduce unnecessary antibi- would be IL-6, IL-8, nCD64, and TNF-, whereas mid
otic use are PCT and nCD64. Use of PCT has been shown phase (1224 hours) would be PCT, and the late phase
to decrease the duration of antimicrobial treatment by 24 (>24 h) being CRP [14]. Based on the data summarized
days, in a meta-analysis of randomized controlled trials in above, diagnostic utility of the following biomarkers
intensive care units, which did include neonates (n = 121), would be most practical in the clinical setting: IL-6, IL-8,
with no apparent adverse clinical outcomes [92]. A pilot TNF-, nCD64, PCT, and CRP. These have been summa-
study using PCT to guide treatment has reported a signi- rized in Table 3. The availability of daily testing and TAT in
cant absolute risk reduction of 27% (P = .002) in neonates clinical laboratories of the listed biomarkers would be
treated for 72 hours, with an average decrease of 22.4 somewhat dependent on the institutional resources, but
hours for the duration of antibiotic therapy [93]. A multi- most centers with Level IV NICUs would be expected to
center, randomized trial on the efcacy and safety of have access to at least 2 or more of the tests (specically,
PCT-guided treatment in neonates is ongoing (clinical nCD64, PCT, and CRP). Regarding the cytokines, a few
trials.gov: NCT00854932) [66]. caveats need to be considered. First, despite a theoretical
As regards nCD64, its high NPV and decrease in concen- TAT ranging 1.56 hours, cytokines are not commonly
tration on sequential measurements in blood culture- measured in a stat laboratory, and therefore their clinical
positive cases on treatment suggest the potential for its use in an emergency setting is unreliable, at least in more
use in deciding the initiation and duration of antibiotic than 90% of all clinical laboratories. Second, cytokines
use [43, 76, 81]. Currently, a single-center randomized con- measurement accuracy is inuenced by the preanalytical
trolled trial using sequential nCD64 concentrations for de- phase: cytokines should be measured after short time
cision making to stop antibiotics early in EOS and LOS in from blood collection; the blood sample should be collect-
neonates is planned (clinicaltrials.gov: NCT01825421). ed in tubes containing specic anticoagulants, and the tem-
perature of sample transport transportation also inuences
the stability of these molecules.
Expert Commentary Among biomarkers of sepsis for which limited or no in-
From the practical viewpoint, 1 approach would be to cat- formation is available on neonates, but based on research
egorize the biomarkers of neonatal sepsis based on the studies conducted on adults and children, those that would
detection time from the perspective of point-of-care diag- have strong potential to be clinically useful are angiopoie-
nostics into early, mid, and late phases [14]. Dening the tin 2, haptoglobin, soluble triggering receptor expressed on
phase is complicated by the fact that it is dependent upon myeloid cell (sTREM) and soluble urokinase plasminogen
when the sample was collected and analyzed during the activator receptor (uPAR) [15, 49, 94100].
course of the neonatal sepsis. Thus, the start point It is imperative that future studies focused on establish-
could be the time when the clinical suspicion of sepsis ing reliable biomarkers for diagnosis of neonatal sepsis
was initially triggered or when the neonate was rst should not only be adequately powered for sample size
brought to the attention of the medical team. In the latter but also use the gold-standard denition of blood-culture
scenario, this may not coincide with the onset of illness and proven pathogen-specic sepsis. The use of minimal

Table 3. Diagnostic Clinical Utility of Selective Biomarkers At Different Phases of Neonatal Sepsis*
Phase Biomarker Cutoff Values Blood/Serum/Plasma Volume Required Turnaround Time
Early (212 h) IL-6 10150 pg/mL 2100 L 1.56 h
IL-8 60300 pg/mL 250 L 1.56 h
TNF- 1290 pg/mL 2200 L 1.56 h
nCD64 2.43.6% 50 L 12 h
Mid (1224 h) PCT 0.55.75 ng/mL 20200 L 20 min5 h
Late (>24 h) CRP 410 mg/L 520 L 14 h
Abbreviations: CRP, C-reactive protein; IL, interleukin; LOS, late-onset sepsis; nCD64, neutrophil CD64; PCT, procalcitonin; TNF, tumor necrosis factor.
*See text for more details.

For EOS.

For LOS.

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242 Bhandari

blood volumes for sample collection and quick TAT for di- Novel biomarkers of neonatal sepsis that require addi-
agnostic tests are always critical factors for the neonatal tional research include MR score, haptoglobin, serum
population. In addition, the utility of serial measurements amyloid A, hepcidin, interalpha inhibitory proteins
of such biomarkers should be assessed for antibiotic stew- (IaIp), MBL, angiopoietin 2, sTREM, and soluble uPAR.
ardship in the neonatal population. The utility of serial measurements of such biomarkers
should be assessed for antibiotic stewardship in newborns.
Renements to allow early, accurate, and cost-effective
Five-Year View
identication of pathogens responsible for neonatal sep-
One would anticipate that given the strength of the evi- sis would be anticipated in the next 5 years.
dence noted in the preceding pages, nCD64 and PCT
would emerge as the best combination of biomarkers for
diagnostic accuracy of neonatal EOS and LOS as well as
Acknowledgments
for deciding upon the initiation and duration of antibiotic
Potential conicts of interest. Author: No reported conicts.
therapy. In nurseries where the access to the above resourc- Author has submitted the ICMJE Form for Disclosure of Potential
es is limited or unavailable, CRP measurements would be Conicts of Interest.
preferred or continue to be used for the same purposes.
In terms of newer biomarkers, those listed in Tables 1
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