NEONATAL SEPSIS

Newer Diagnostic markers

 Neonatal Sepsis
• Pathophysiology
• Classification & Epidemiology
• Investigations
– Traditional approach to investigations
– Newer Biomarkers
– Gold standard
 Neonatal sepsis

Sepsis is a syndrome in which there are clinical

signs of infection, Presence of bacteremia or
positive culture from otherwise sterile site

Fanaroff 10 th Ed.
Pathophysiology
Elevation of bio-markers in sepsis
Epidemiological Classification of Neonatal
Sepsis
Characteristic Early Late
Time of onset Birth to 72 hours >72 hours
Complications of + ±
pregnancy or delivery
Source of organism Maternal genital tract Maternal genital tract,
postnatal enviornment

Usual clinical Multisystem Focal

presentation Pneumonia frequent Meningitis frequent
Mode of transmission Vertical, in utero, or Nosocomial, horizontal
intrapartum

Fanaroff 10 th Ed.
 Early Onset Sepsis
• Mode of infection: vertical transmission (ascending amniotic fluid
infection (T. pallidum and L.monocytogenes cross placenta) or through
acquisition of bacterial flora from mother’s anogenital tract during
vaginal delivery (GBS and Gram negative enteric bacilli).

• Developed nations (USA):

– MC causative bacteria are GBS (38-43%) and E. coli (24-29%).
– Overall incidence 0.98 per 1000 live births (< 1500g 10.96 per 1000 live births,
1500-2500 g, 1.38 per 1000 LB).
• India:
– NNPD data: incidence of neonatal sepsis - 30 per 1000 live births; EONS -50%
– DeNIS study 2011-2014, incidence of sepsis is 21.8 per 1000 live births; EONS-
68%; case fatality rate- 26% Incidence is 7 times higher in preterm, 20 times
higher in VLBW Overall case fatality rate of sepsis overall is around 11 to 16 %.
 EONS risk factors
• Preterm
• Low birth weight (attack rate 5.99/1000, 2.51/1000, 0.89/1000
live births for infants with BW <1500,1500-2500 and >2500g)
• Clinical chorioamnionitis (4-10times)
• Prolonged ROM ≥18 hrs (2-10 times)
• Prolonged labour ≥24 hrs
• Foul smelling liquor (2-3 times)
• Single Unclean or ≥3 sterile examinations
• Maternal fever with evidence of bacterial inf. 2 weeks prior
to delivery
• Perinatal asphyxia (≤6 @ 5min.)
• Triple I (2-3.5 fold)

Indian Pediatr 1974

Fanaroff 10 th Ed. EONS.Clinical microbiology Rev. 2014 J Trop Pediatr 1994
 EONS risk factors
• Exposure to Triple I increases the risk of adverse outcome by
2- to 3.5-fold, independent of the duration of infection.
• Adverse fetal/neonatal outcomes include: perinatal death,
asphyxia,EOS, septic shock, pneumonia, meningitis, IVH,
cerebral white matter damage, and long-term disability
including cerebral palsy, as well as morbidity related to
preterm birth.
• Overall, Triple I is associated with up to 40 percent of cases
of early-onset neonatal sepsis.
 EONS risk factors-India

Study by Dutta S et al 2009 (PGI Chandigarh): 601 enrolled, 13 risk factors assessed.

Score of 0-6 –Careful monitoring for 72 hrs

Score of ≥7 – Take blood culture & start prophylactic empirical antibiotics.
 EOS risk calculators
• EOS calculators (Kaiser Permanente) Kuzniewicz et al
JAMA 2017
– used to estimate the risk of EOS based on risk factors (eg,
newborn clinical condition, highest intrapartum maternal
temperature, maternal group B Streptococcus [GBS] status,
the administration of maternal IAP, gestational age, duration
of rupture of membranes).
– May help guide decision-making regarding diagnostic
evaluation and empiric antibiotic treatment.
– EOS risk calculators are not valid for preterm infants (<34
weeks gestation), and they do not apply to late-onset sepsis.
 EOS risk calculators
• Implementation of sepsis calculator associated with reduced
antibiotic usage [OR = 0.22 (0.14–0.36); NNT 22], laboratory
tests [OR = 0.14 (0.08–0.27), NNT = 8], and admissions to
neonatal unit [OR = 0.24 (0.11–0.51), NNT = 7]

• There was no difference in mortality, culture positive EOS, and

readmissions.

Systematic review by Deshmukh et al 2019

 Late Onset Sepsis
• Acquisition of infection is predominantly through the infant’s
environment.
• Common organisms: CONS and S. aureus, invasive candidiasis,
GBS and E. coli.
• Escherichia coli is frequently a cause of urosepsis in young
infants.

• Risk factors: Extreme prematurity is one of the greatest risk

factors for late-onset sepsis.
– Others: Prematurity, NICU admission, use of H2 blockers andPPIs,
gastrointestinal tract pathology, mechanical ventilation, invasive
procedures, administration of parenteral fluids
 LONS risk factors
Anatomic Defects
• Obstructive uropathy
• Gastroschisis
• Myelodysplasia

NICU Care/Environmental Issues

• Frequent invasive procedures
• Poor skin integrity
• Repeated courses of antibiotic
therapy
• Indwelling vascular catheters
• Long-term MV
• Long-term glucocorticoid therapy
• NICU understaffing and impact
On Handwashing
Poor Nutrition
• Delayed initiation of enteral feedings
• Prolonged period to reach full
enteral feedings
• Prolonged period of time to regain
birth weight
 Working classification of sepsis

• Symptoms/signs +, • Symptoms/signs +, • Symptoms/signs +

investigations - investigations + investigations +
cultures - cultures - cultures +

Suspect Probable Definite or

sepsis sepsis proven sepsis

PEDIATRICS Vol. 102 No. 4 October 1998

 Diagnosis of sepsis
• Ideal biomarker:
– Stable compound
– Adequate time window for specimen sampling (sustained
increase or decrease in level for at least 48 h after the onset
of clinical manifestations)
– Quantitative measurement
– Small volume of specimen
– Easy method of measurement
– Quick lab turnover time
– Results comparable between labs
– Low cost
Diagnosis of sepsis
 Blood culture: Gold standard

• In EONS - positive in 20 %
• Procedural issues:
– NNF - Single blood culture of atleast 0.5 ml blood for
pediatric culture bottle
– AAP - 1 ml of blood for pediatric culture because 0.5 ml
- cannot detect low level bacteraemia (4 CFU or less)
and 25 % of infants with sepsis have low bacteraemia.
– Use of umbilical venous catheter, indwelling arterial
catheter or capillary sample – Increase risk of
contamination
 Blood culture: Gold standard
• Bac T/Alert system for detection of
bacteremia :
• Prospective study from India, 101 cases with
suspected sepsis enrolled
• Mean time to positivity with manual & Bac T/
Alert - 53.1 and 14.3 hrs (P- <0.001)
• Equally efficacious in correctly identifying a
bacterial growth in a significantly shorter time
Traditional diagnostic markers of Neonatal
sepsis

Septic screen:
– total leukocyte count (TLC)
– absolute neutrophil count (ANC)
– immature to total (IT) neutrophil ratio
– micro-erythrocyte sedimentation rate
– C reactive protein (CRP)
 Sepsis Screen
COMPONENTS ABNORMAL VALUES

Total Leukocyte count (TLC) <5000/mm3

Absolute Neutrophil Count (ANC) As per Manroe’s chart for Term and
Mouzhino’s chart for VLBW

I:T ratio >0.2 in PT and >0.27 in T

Micro-ESR >15 in first hour

CRP >10mg/L
 Sepsis Screen
• Presence of two abnormal parameters in a screen is
associated with a sensitivity of 95-100%, specificity
of 83%, positive and negative predictive values of
27% and 100% respectively in detecting sepsis

• If ≥2 parameters are abnormal, it should be

considered as a positive screen

• If the screen is negative but clinical suspicion

persists, it should be repeated 12-24 hours apart
 Sepsis Screen
• Test result will not affect the decision to start
treatment if there is strong clinical evidence of
the infection
• Test is most useful when history and
examination of the patient leave a serious
doubt about the presence of infection.
• Sepsis screen is not that useful in ruling in
sepsis.
 Sepsis Screen-Individual parameters
Test Sensitivity NPV False positive False negative

TLC 17-90% 91 Crying baby, MAS, PIH, IVH,

pneumothorax hemolytic disease

ANC 38-96 96-99 Stressful labor, seizures -do-

hypoglycemia

I/T ratio 60-90 99-100 Maternal Variable

fever,oxytocin,stress
labor,IVH,seizures

CRP 47-100 71-99 Bacterial First 12 hrs, early

contamination, fungal onset sepsis,
infection, overwhelming
RDS,asphyxia,lipemia, sepsis
hemolytic serum

Micro ESR 27-50 94-97 Anemia DIC

 CRP
• Most studied biomarker in sepsis
• IL-6 released by granulocytes, stimulates liver to produce CRP
• Serum levels increase upto 100- 1000 times
• Begins to ↑ at 6 to 8 hrs, peaks at 2- 3 days
• Elevation of CRP in neonate always thought to be de novo,
since very little passes through placenta.
• Role for early diagnosis is limited
• False positive: Viral infection, ischemia, hemolysis, meconium
aspiration, chorioamnionitis without bacterial invasion, IVH
• In combination with other markers (IL6, PCT, IL8, etc.),
sensitivity improves to 90-100%
 CRP
• CRP in LOS Brown et al Cochrane 2019: 20 studies,
1615 neonates
– Sensitivity 0.69 (95% CI 0.51 to 0.82)
– Specificity 0.80 (95% CI 0.69 to 0.87)
– Positive likelihood ratio 3.37 (95% CI 2.26 to 5.03)
– Negative likelihood ratio 0.39 (95% CI 0.24 to 0.63)
• CRP threshold 5-10 mg/L
• Measurement :
– Bed side : Visual agglutination
– Lab : Immuno - turbidometry
 CRP
Advantages:
• Useful negative predictor (high NPV esp. after 24
hours of symptoms)
• Small amount of blood
• Rapid results
• Cheap
• Serial levels for monitoring response can be done
 CRP
Limitations:
• Non specific marker of inflammation.
• Cannot differentiate viral or bacterial origin of disease.
• EOS: Levels may continue to rise till 48 hours- cannot
guide appropriate antibiotics coverage.
• Late marker with low sensitivity at onset of infection.
• Not much evidence to determine duration of therapy by
monitoring normalization
• Value of high in NVD and Em LSCS
• Preterm values are less as compare to term
 Blood culture
• It is the gold standard for diagnosis of neonatal
sepsis and should be performed in all episodes
of sepsis prior to starting antibiotics

• A positive blood culture along with the

sensitivity pattern of the isolated organism is
the best guide to antimicrobial therapy

Remington and Klein 8th Ed.

 Contd.

• In 20% of suspected EONS and 30% of suspected

LONS, blood culture is positive
• Single site culture with blood volume of ≥1 mL

is sufficient to document sepsis in neonates.

• To reach laboratory within 4 hours of innoculation
• Not to be taken from indwelling central lines but can be
taken from freshly placed central lines
Remington and Klein 8th Ed.
NEWER BIOMARKERS FOR DIAGNOSIS
Timeline of elevation of acute phase
reactants in neonatal sepsis
Timeline of Elevation of bio-markers in
sepsis
Procalcitonin
 PCT
• Formed by Monocytes, hepatocytes and nephrons.
• 116 aminoacid hormokine
• Also Produced by C-cells of thyroid gland as a pro-
hormone of calcitonin.
• Kinetics
– Rise: 4-6 hours
– Peak: 24 hours
– Decline: Rapid, half every 1-1.5 days
– t1/2: 24-35 hours
 PCT
• Meta- analysis (Vouloumanou et al 2011): 16 studies;1959 neonates,
single point procalcitonin at presentation, Culture proven or clinically
diagnosed sepsis
– Sensitivity: 81% (74–87%) [EOS-76% vs LOS 90%]
– Specificity: 79% (69–87%) [EOS-76% vs LOS 88%]
– AUC: 0.87.
– Better diagnostic accuracy for LOS vs EOS (AUC for LOS: 0.95 vs 0.78 in LOS)
– Statistical heterogeneity
• Meta analysis by Pontrelli et al 2017: Diagnostic accuracy of PCT
– 17 studies,1408 patients (1086 neonates and 322 children). Studies on neonates
with EOS/LOS
– Sn 0.85 (0.76; 0.90) and specificity of 0.54, CI (0.38; 0.70) at the PCT cut-off of
2.0-2.5 ng/ml. PCT shows a moderate accuracy for the diagnosis of sepsis in
neonates with suspected sepsis at the cut-off of2.0-2.5 ng/ml.
 PCT
• NeoPINS study (Lancet 2017 Stocker et al): Procalcitonin-
guided decision making for duration of antibiotic therapy in
neonates with suspected early-onset sepsis: a multicentre,
randomised controlled trial
– 1710 neonates enrolled with suspect EOS >34 wks GA; randomly
assigned to either procalcitonin-guided therapy (n=866) or standard
therapy (n=844)
– Procalcitonin group: Duration of antibiotic therapy was reduced No
sepsis-related deaths occurred, and 9 (<1%) of 1710 neonates had
possible re-infection.
– Procalcitonin-guided decision making was superior to standard care in
reducing antibiotic therapy in neonates with suspected early-onset
sepsis
PCT
 PCT
• False positive
– Maternal pre-eclampsia
– Respiratory distress syndrome
– Hemodynamic instability, asphyxia
– Pneumothorax
– Intracranial haemorrhage
– Trauma and surgery
– Post resuscitation/shock
• False negatives:
– Localized infection
– Atypical bacteria- chlamydia, mycoplasma
– Drawn too early in course
 PCT vs CRP
• Advantages
– Rapid rise in response to infection- useful in early diagnosis
– Decline in levels with control of infection
– Half life – 24 hrs
– Levels not influenced by viral infection
– Correlates with severity of infection
– Yu et al (Metanalysis of 4 studies):Pooled sn significantly higher for
PCT compared to CRP (72% vs 55%)
• Limitations
– Specific cut-offs required for various gestational & early postnatal age
– Higher cost: Limits routine serial monitoring
– Not enough evidence to recommend using as single marker.
– Bedside non availability
 Newer Diagnostic Markers
• Serum amyloid A protein
– Produced mainly in liver, also by macrophages and
endothelial cells
– Anti inflammatory function
– Rises by 3-4h; peaks by 2-3 days; returns baseline
5-7d
– Better diagnostic accuracy for EONS than CRP-
Sn(96 vs 30%), sp(95 vs 98%)
 Newer Diagnostic Markers
• Mannose binding lectin
– Pattern recognition molecule in lectin complement
pathway
– Activates complement system
– Cutoff point of ≤0.93 μg/mL to differentiate EONS
and non EONS
– Sn 76%, sp 56%, PPV 60%, NPV 67%
 Newer Diagnostic Markers
• Liposaccharide binding protein
– LBP are synthesized in liver
– Peak elevation by 6- 12 hrs and persists for 72 hrs
– Sn(92%) and Sp(86 %)
– Highly elevated levels in early onset sepsis
– Prolonged time period to identify sepsis
 Newer Diagnostic Markers
• IL 6
– enhances acute phase proteins release by liver and causes
SIRS
– Very early marker
– High sensitivity (89%) and NPV(91%) at onset
– Umbilical cord levels for EONS- high accuracy (Sn- 87-100%;
NPV 93-100%)
– Pooled sn and sp from meta anlysis is 79% and 84%
– Accuracy improves in combination with CRP
– Limitations: Short half life and varying cut off (0.15 – 1.35
ng/ml)
 Newer Diagnostic Markers
• IL 8
– Attract PMNs and monocytes to the site of
inflammmation
– High accuracy in both EOS and LOS
– Sn - 80-91%; Sp- 76-100%
– Limitations: Short half life and varying cut off (0.5-
70 ng/ml)
 Newer Diagnostic Markers
• IL10, TGF β
– Helps in downregulation of immune reponse
– Role in diagnosis of EONS is not yet established
– No major studies available
 Newer Diagnostic Markers
• Neutrophil CD64; neutrophil/ monocyte CD11b
– Detected by flow cytometry
– Advantages: Minimal blood volume(50μl)
– Results in 4 hrs
– Wide diagnostic window ( expressed for 24hrs)
– High sensitivity and specificity (CD 11b mainly for
EONS)
– Limitations: high cost & limited availability ,Meta
analysis of n CD64 shows Sn and Sp(< 80%),not
satisfactory marker in diagnosis of sepsis
 Genomics

Molecular diagnosis
1. Whole blood tested directly by target amplification:
Advantage of rapid diagnosis but the challenges of suboptimal sensitivity and specificity.
Universal 16S ribosomal DNA (rDNA) target present in nearly all bacteria but not in
human cells
2. Pre enrichment of whole blood before target amplification: Whole blood
was incubated in tryptic soy broth (TSB) for up to 5 hours before nucleic acid
extraction and PCR; high level of overall agreement (96.8%), specificity
(97.5%), and negative predictive value (99.2%), but also revealed the inability
of PCR to detect several culture-proved cases of EOS in these infants.
3. Fluids from positive blood culture bottles tested by polymerase chain
reaction (PCR): As little as 0.1 mL of fluid was removed from positive blood
culture bottles, and nucleic acid extraction and PCR or NASBA target
amplification were performed.
 Genomics

• PCR advantages over blood culture:

– detection of DNA from dead or intracellular
bacteria, not detected by culture.
– identification of novel or emerging organisms
associated with BSI that are uncultivable or
fastidious in nature.
 Genomics

4. Nucleic acid sequence-based amplification (NASBA):

– NASBA is an isothermal nucleic acid amplification assay
that targets single-stranded templates so it preferentially
amplifies RNA targets, and thus has an advantage over PCR
in that ribosomal RNA (rRNA) genes are expressed at much
higher levels at thousands of copies per cell.
– Sensitivity and specificity for gram-positive and gram-
negative bacteria is 96.1% and 98.6% and 100% and 95.9%,
respectively, and found 100% sensitivity and specificity for
Candida.
 Genomics

5. PCR in conjunction with sequencing or microarray analysis:

– Cleven and colleagues developed a DNA microarray that directly
identified 3 common bloodstream pathogens (Staph. aureus, E coli,
an P. aeruginosa) from positive blood culture bottles without prior
nucleic acid target amplification.
– The array contained recombinant plasmid-based species-specific
probes, 200 to 800 base pairs in length, that targeted housekeeping
genes, virulence factors, and antibiotic resistance genes.A high level
of correlation was found between the microarray analysis and
conventional phenotypic identification for all 3 pathogens.
Antibiotic susceptibility testing showed perfect correlation for S
aureus, with slightly lower correlations for antibiotic resistance
genesin E coli and P aeruginosa.
 Genomics

6. Nonamplification- based fluorescence in situ

hybridization (FISH):
– FISH on positive blood culture fluids. FISH probes
against variable regions of the 16S rRNA to
identify Streptococcus spp, E faecalis, S
aureus,CONS, E coli, P aeruginosa,
Enterobacteriaceae family.
– Studies have shown 98-100% Sn and 99% Sp.
 Genomics

7. Matrix-assisted laser desorption ionization time-of-flightmass

spectrometry (MALDI-TOF MS)
– accurate results for BSI with single pathogens from positive blood
culture bottles in about1 hour’s time
– On the basis of detection of common housekeeping and ribosomal
proteins, MALDI-TOF MS technology has been shown to accurately
identify bacteria, yeast, filamentous fungi, Nocardia, and mycobacteria,
often at reduced costs compared to traditional identification methods.
– pathogen identification has been shown to occur several days earlier,
resulting in reduced time until effective antimicrobial therapy is
administered.
– An additional benefit of this technology is that it can detect
antimicrobial resistance.
 Proteomics

• Separates the proteins from complex compound and

identifies the proteins of interest; Separation by molecular
weight, isoelectric point, hydrophobicity, affinity
– Protein identification by digesting them into peptides
– Proteomics biomarker for diagnosis of chorioamnionitis and
EONS
– 4 biomarkers in amniotic fluid and MR (Mass restricted) score
generated- neutrophil defensin -1 and 2, S100A12, A100A8
– MR score 3-4 - highest in detecting intra amniotic infection
and hence predicting EONS
 Metabolomics
• Estimation of entire metabolome of various biochemical pathways
(in urine or plasma), few studies reported.
– Sepsis induces hypoxia, oxidative stress, and an increased demand for
energy resulting in both glucose metabolism and oxidative metabolism of
fatty acids; necessitating the monitoring of metabolome dysregulations
during neonatal sepsis.
– Metabolomics can characterize thousands of intermediate to low
molecular-weight carbohydrates, amino acids, lipids and other molecules
generated by the interaction between host genome, the gut microbiome
and environment. It is thus a useful tool to investigate metabolic
perturbations related to neonatal sepsis for identification of novel
biomarkers.
– Useful in early diagnosis of infection(preclinical stage)
– In future bedside urine dipsticks can identify metabolites
 Blood culture
• It is the gold standard for diagnosis of neonatal
sepsis and should be performed in all episodes
of sepsis prior to starting antibiotics

• A positive blood culture along with the

sensitivity pattern of the isolated organism is
the best guide to antimicrobial therapy

Remington and Klein 8th Ed.

 Contd.

• In 20% of suspected EONS and 30% of suspected

LONS, blood culture is positive
• Single site culture with blood volume of ≥1 mL

is sufficient to document sepsis in neonates.

• To reach laboratory within 4 hours of innoculation
• Not to be taken from indwelling central lines but can be
taken from freshly placed central lines
Remington and Klein 8th Ed.
 Summary
• Current (traditional) approaches to diagnosis are too simplistic and imprecise to identify
affected individuals accurately.

• Nature of neonatal medicine, with its highly intensive and frequent monitoring of infants,
often over protracted periods and from birth, makes it ideal for translation to the
precision medicine setting.

• Allows a more predictive and preventive approach, where septic infants can be identified
ahead of clinical disease phenotype expression, ensuring prompt and effective antibiotic
use.

• The era of precision medicine for neonatal sepsis, built on the platforms of omics
technologies, holds promising potential to ensure quicker and more accurate diagnosis of
neonatal sepsis and thus personalized treatment and improve prognosis of septic
neonates.
 LUMBAR PUNCTURE
• In EOS, it is indicated in the presence of a
positive blood culture or if the clinical picture is
consistent with septicemia or meningitis
• It is not indicated if antibiotics have been
started solely due to the presence of risk factors
(clinical chorioamnionitis, ≥3 risk factors)
• In case of late onset sepsis, it should be done in
all prior to starting antibiotics
THANK YOU