Laboratory Diagnosis

in Newborn Infectious
Disease
& Neonatal Sepsis
Clinical Pathology Department,
Faculty of Medicine Universitas Gadjah Mada /
Dr. Sardjito Hospital

SCCM Consensus Definition

Infection

SCCM Consensus Definition

Sepsis
Infection, plus
2 or more SIRS criteria

SCCM Consensus Definition

Severe Sepsis

SCCM Consensus Definition

Septic Shock

Parasite

SIRS

Others

Virus

Infection

Severe Sepsis
Sepsis

Fungus

Multi
shock Organ
Dysfunction
syndrome

Bacteria BSI
Adapted from SCCM/ACCP Consensus Guidelines

Burns

Trauma
Pancreatitis

Greg Martin, David mennino,et al.

N Engl J Med, vol 347, No 13 Sept 26, 2002 www.nejm.org

Mortality rate - increases with increased severity

7%
16%
20%
46%

in
in
in
in

patients
patients
patients
patients

with
with
with
with

SIRS
Sepsis
Severe Sepsis
Septic Shock

Rangel-Frausto, et al (JAMA 1995)

Neonatal mortality ASEAN countries

Life birth
(000)

Perinatal
mortality

Early
neonatal
death

Neonatal
mortality

Singapore

47

Malaysia

549

Thailand

1082

20

13

Philippines

2029

23

12

15

Vietnam

1593

37

13

15

Indonesia

4564

30

14

18

Laos

195

57

26

35

Cambodia

461

66

31

40

Myanmar

1194

65

30

40

Country

Brunei

WHO Data : Neonatal and perinatal mortality : country, regional and global estimates. 2006

Cause of neonatal death in SEA

Neonatal Major Infection

Infection

No. of cases

Case Fatality
Rate (%)

No. of Death

Acute Lung
Inf

2.500.000

30

750.000

Tetanus
neonatal

438.000

85

372.000

Sepsis

750.000

40

300.000

Diarhea

25.000.000

0.6

150.000

Meningitis

126.000

40

50.400

Stoll BJ : The global impact of infection Clin Pernatol

1997; 24:1-21

Diagnostic criteria for sepsis

Infection, documented or suspected, and some of the
following:
General variables
Fever (> 38.3 C)
Hypothermia (< 36 C)
Tachycardia (heart rate > 90/min, or >2 SD above the normal
value for age)
Tachypnea (increased respiratory rate)
Altered mental status
Significant edema or positive fluid balance (> 20 mL/kg over
24 hrs)
Hyperglycemia (plasma glucose >120 mg/dL or 7.7 mmol/L) in
the absence of diabetes

ACCP/SCCM Consensus Definition, Crit Care Med 2003

Inflammatory variables
Leukocytosis (WBC count > 12,000/uL)
Leukopenia (WBC count < 4,000/uL)
Normal WBC count with > 10% immature forms
Elevated plasma C-reactive protein (CRP)
Elevated plasma procalcitonin (PCT)

Hemodynamic variables
Arterial hypotension (SBP < 90 mmHg, MAP < 70, or an
SBP decrease > 40 mmHg in adults or < 2 SD below
normal for age)
SvO2 > 70%
Cardiac Index > 3,5 L*min-1M-23

ACCP/SCCM Consensus Definition, Crit Care Med 2003

Organ dysfunction variables

Arterial hypoxemia
Acute oliguria (reduced urine output)
Creatine increase
Coagulation abnormalities (elevated D-dimer, prolonged
PT, reduced protein C)
Ileus (absent bowel sound)
Thrombocytopenia (platelet count < 100,000/uL)
Hyperbilirubinemia

Tissue perfusion variables

Hyperlactatemia (> 1 mmol/L)
Decreased capillary refill or mottling

ACCP/SCCM Consensus Definition, Crit Care Med 2003

2005

2005

New Perspectives and Techniques

in the Diagnosis of Infection and Sepsis
Clinical
Features

Fever
Shock

Classically
Laboratory
Parameters

CRP
ESR
D-dimer
Fibrinogen
Leucocytosis
Bacteremia

New Laboratory
Parameters

Hypo
phosphatemia

New Techniques

PCR
(light cycler)
Oligo array
technique

Cytokine
Procalcitonin

Identification and
characterization
Bacteria cytokine
Inflammation
marker

IMAJ, vol 3, Juni 2001

Sepsis - pathophysiology

PATHOGEN
Gram positive

Gram negative

Response of the human organism to

microbe invasion, the outcome depends on
the interrelationship of pro and antiinflammatory cytokines.
If the infectious source can be controlled
and the function of pro and antiinflammatory cytokines is balanced
survives.
If the function of pro-inflammatory
cytokines for any reason-outweighs the
anti-inflammatory system, the developing
organ damage becomes irreversible and
the patient dies.

Sir William Osler 1904

The Evolution of Modern Medicine

Diagnosis of sepsis

There is no specific pathognomonic clinical

or laboratory parameter of sepsis

Early detection and timely therapeutic

intervention is crucial for improved
outcome of patients with sepsis

Early diagnosis of sepsis is key for

improved survival

The diagnosis of sepsis is difficult and not

reliable based on general signs and
symptoms such as fever, tachycardia and
tachypnea

Infectious agent
Laboratory tests important in identifying the infectious agent

In one recent, prospective study, positive

blood cultures were found in:

17% of patients with sepsis,

25% with severe sepsis,
69% with septic shock.

Two major reasons:

The patients are indeed septic with

bacteremia but the organisms did not
grow under normal circumstances in the
culture medium

The septic state may have resulted not

from bacteremia but from pyrogenic
cytokine activation

Gram-negative organisms the most

common isolates from blood cultures

Escherichia coli,
Klebsiella pneumoniae,
Enterobacter cloacae,

the epidemiology of sepsis has changed

in the
last 2 decades gram positive

Gram-positive organisms accounted for

more than 50% of all positive blood
cultures from septic patients

Coagulase-negative staphylococci,
Staphylococcus aureus,
Enterococcus faecalis

Several reasons for the increasing number

of Gram-positive infections:

Selection of resistant Gram-positive organisms

from empiric antibiotic therapy directed at
Gram-negative infections,
Increased use of vascular access catheters and
implantable devices/prostheses,
Shared antibiotic resistance between Grampositive organisms, and increased virulence of
Gram-positive pathogens.

because of advances in medical

technology

Microbial factors in sepsis

Gram-negative bacteria

Cohen, Nature: 2002 420:885

What microorganisms cause severe

infections and sepsis in babies?
Prenatal

During Delivery

After Birth

Rubella (German
measles)

Group B
streptococcus
(GBS)

Respiratory
syncytial virus
(RSV)

Cytomegalovirus
(CMV)

E. Coli

Candida

Varicella-zoster virus
(chickenpox virus)

Herpes
simplex virus

Haemophilus influe
nzae type b
(Hib)
Enterovirus

Listeria
monocytogenes

Laboratory Indicators of Infection /

Sepsis
Laboratory Test

Findings

Interpretation

White blood cell

count

Leukocytosis or
leukopenia

Endotoxemia may
cause early
leukopenia

Platelet count

Thrombocytosis or
thrombocytopenia

High value early

may be seen as
acute-phase
response; low
platelet counts
seen in overt DIC

Coagulation
cascade

Protein C
deficiency;
antithrombin
deficiency;
elevated D-dimer
level; prolonged PT
and PTT

Abnormalities can
be observed before
onset of organ
failure and without
frank bleeding.

Creatinine level

Elevated from
baseline

Doublingindicates
acute renal injury

Laboratory Test

Findings

Interpretation

Lactic acid level

Lactic acid > 4

mmol/L (36 mg/dL)

Indicates tissue
hypoxia

Liver enzyme levels

Elevated alkaline
phosphatase, AST,
ALT, bilirubin levels

Indicates acute
hepatocellular injury
caused by
hypoperfusion

Serum phosphate
level

Hypophosphatemia

Inversely correlated
with
proinflammatory
cytokine levels
(TNF, IL-6, IL-2
receptors)

C-reactive protein
(CRP) level

Elevated

Acute-phase
response

Procalcitonin level

Elevated

Differentiates
infectious SIRS from
noninfectious SIRS

What is expected from such a

marker ??

Fast increase to be present at the onset

or even before the appearance of the
clinical signs of infection/sepsis

To be highly sensitive and specific for

infection/ sepsis (differentiation between
infectious and non infectious causes of
inflammation, organ dysfunction and
shock)

Improve accuracy of clinical diagnosis

To indicate the effectiveness of therapy

Recent Laboratory Marker :

1.
2.
3.

Existing Marker: C-Reactive Protein (CRP)

Emerging Marker: Procalcitonin (PCT)
Promising Markers:
- Interleukin 6 (IL-6)
- Protein membran : CD 64, CD 11b
- Circulating protein:
Neopterin, Endotoksin, protein HMGB 1 (high-mobility
group-box 1), NT-proBNP (N-terminal pro-brain natriuretic
peptide)

C-REACTIVE PROTEIN (CRP): (1)

Introduced in 1930, analitycal methode has
developed since 1947 as infection marker
Technology high sensitive & ultra sensitive
has analitycal sensitivity: 0,07 ug/mL (mg/L)
Property (1):
MW: 120-140 kDalton, 206 peptida, can not
pass the barrier placenta
Produced in liver trigering by IL6, IL1, TNF,
TGF, IFN

C-REACTIVE PROTEIN (CRP): (2)

Property (2):
Recognation & activation capability may
recognize foreign pathogen & material of
damage cell cleansing agent
An phase acute protein : increase in 4 hr up to
the peak level in 24 -72 hr
Persisten increasing in 1 week indicates a
treatment failure or disease comorbidity

What is PCT??

PCT is the prohormone of the hormone

calcitonin but are distinct proteins

Calcitonin is exclusively produced by Ccells of the thyroid gland in response to

hormonal stimuli, whereas

PCT can be produced by several cell types

and many organs in response to proinflammatory stimuli, in particular by
bacterial products

PROCALCITONIN (PCT):
History:
Introduced in 1984 as a prohormon protein
consisted of 116 asam amino, MW 13-14
kDalton
Era 1990 : developed as a marker of bacterial
infection
Property (1):
Production is stimulated by the presentation of
endotoksin, increase in 2 hr, peak in 12-24 hr,
decrease slowly in 36-72 hr

PROCALCITONIN (PCT): (2)

Property (2):
Produced in C cell of tiroid gland under IL6,
IL8, TNF regulation
Normal condition : all PCT cleavaged into
calcitonin
Infection : expression of gena Calc-1 will
stimulate all types of parenchym tissue cells to
release PCT

PROCALCITONIN (PCT): (3)

Properti 3:
As secundary inflammation mediator inhibit
prostaglandin & tromboksan sintesis at
limfosit
Increasing level depands on the stage of sepsis
Normal: < 0,05 ng/mL (ug/L), in bacterial
infection > 2 ng/mL

PCT reference ranges

and Interpretation of PCT
serum or plasma levels

Performance PCT
Referens

Level

Bender, et al,
2008 (Neonatal
Setting)

> 2.5 ng/mL

(+ IL 6 > 250
pg/mL)

Gaini, et al, 2007

(Community
Setting)
Andreola, et al,
2007
(Emergency
Setting)

2,19 ng/mL

> 2 ng /mL

Sensitivity
71 %

80,7 %

37,7 58,4 %

Spesivicity
88 %

67,8%

93,8 - 93,2 %

INTERLEUKIN 6 (IL-6):

Citokine proinflamatory, reach the peak level

in 3 hr & decrease to baseline level in 8 hr
Increasing of PCT level indirectly influenced
by IL-6, similar with CRP & other acute phase
protein
Methode : ELISA with detection limit 2 4,6
pg/mL

ENDOTOXIN

Lipopolysacharide, component of cell wall of

Negative Gram Bacillus
Strong trigger toward sepsis cascade
Detection:
1. LAL (Limulus Amoebicyte Lysat): very
sensitive, reliabel marker, with gelatin LAL
dan chromogenic LAL
2. EAA (Endotoxin Activity Assay): methode
of Chemiluminesent. Level > 50 pg/mL --- >
marker of negative gram bacillus infection

Protein HMGB 1:

Cromosomal nuklear protein, late onset

proinflamatory cytokine. Secreted by
makrofag culture which stimulated by
endotoxin
Detection limit : 0,6 pg/mL (ELISA)

NEOPTERIN:
Derivat pteridin in monosit/makrofag culture
Mareker of infection (viral & bacterial)
NT-proBNP:
Differentiate : survivor vs non-survivor

CD 11b:

In granula of granulocyt citoplasmic --- >

excessive activation will be mobilized to cell
surface (Upregulation)
Marker for netrofil & monosit, detected by
flowcytometric

CD 64:

Fc-gamma receptor I, function to integrate the

innate & adaptive immune respons infection
may up-regulation
Marker netrofil, detected by flowcytometric
Sistemik infection exspressi >2000

COMPARISON SEVERAL MARKERS

ESR

CBC

CRP

PCT

Whole
blood
(EDTA)

Serum/
Plasma

Serum/
Plasma

Specimen

Whole
blood
(sitras)

Methode

indirect
Direct
(fibrinogen) manual
otomatic

Direct
manual
otomatic

Direct
otomatic

Increasing
Pattern

Slowly,
back to
normal in
weeks

Quick,
much
influenced
by lots
conditions

Quick,
back to
normal up
to no
stimuli

Quick,
back to
normal up
to no
stimuli

Peak level vs
normal

4x

1,5-3 x

15-30 x

> 40 > 200 x

Sepsis

ESR

CBC

CRP

PCT

Confounder:
- sex
- age
- Plasma Protein
-Eritrosit
- etc

+
+
+
+
>>>

X
+
X
X
>>>

X
X
X
X
Tissue
necrosis

X
X
X
X
Ca Tiroid,
Plasmodium
Falciparum

Analitical
Perform:
-Sensitivity
-Spesifisity
-Precisi
-Analytical time

Moderat
Moderat
Moderat
60 mnt

High
Moderat
High
10-60
mnt

High
High
High
< 30 mnt

High
High
High
< 30 mnt

Cost

low

relative

relative

high

Thank You