Neonatal sepsis is a clinical syndrome of bacteremia and systemic infection in infants under 4 weeks of age. It is classified as early onset (less than 72 hours) or late onset (more than 72 hours). Early onset sepsis is usually acquired from the mother during birth and has a sudden and fulminant presentation, while late onset sepsis is often acquired in the NICU from healthcare exposures and other infants. Blood culture is the gold standard for diagnosis but has low sensitivity, while urine and CSF cultures may also be obtained depending on the clinical scenario. Proper collection and handling of specimens is important for optimizing diagnostic yield.

2. Neonatal Sepsis
 Overall incidence = 1-5 per 1000 live births.
 Incidence much higher for VLBW babies :
Early onset sepsis rate = 2%
Late onset sepsis rate = 36%
 The mortality rate is high (13 – 25%) ; higher rates seen in
VLBW babies and in those with early fulminant sepsis.
Data from National Institute of child health and Human Development Neonatal research network
(NICHD-NRN)

3. Neonatal Sepsis
 Neonatal sepsis is defined as a clinical syndrome of
bacteremia with systemic signs and symptoms of infection
in the first 4 weeks of life.
Neonatal sepsis is classified into 2 types depending on the
onset of symptoms.
Early onset sepsis
( < 72 Hrs)
Late onset sepsis
( > 72 Hrs)

4. Early onset sepsis
 Presents in the first 72 hrs of life and is usually a
multisystem fulminant illness with prominent respiratory
symptoms.
 Organism is acquired during antepartum or intrapartum
period from the maternal genital tract.
 Primary sites of colonization includes skin, nasopharynx,
oropharynx, conjunctiva and umbilical cord.

5. Early onset sepsis
 Early onset disease is characterized by a sudden onset
and fulminant course that can rapidly progress to septic
shock and death.
 Organisms most commonly implicated are :
o B- streptococcus (GBS)
o E. Coli
o Staphylocoocus
o Enterococci
o Others like Listeria monocytogenes, Haemophilus influenza,
Streptococcus pneumoniae.

6. Early onset sepsis
 Early onset disease is characterized by a sudden onset
and fulminant course that can rapidly progress to septic
shock and death.
 Organisms most commonly implicated are :
o B- streptococcus (GBS)
o E. Coli
o Staphylocoocus
o Enterococci
o Others like Listeria monocytogenes, Haemophilus influenza,
Streptococcus pneumoniae.

7. Late onset sepsis
 Late onset sepsis is often more insiduous but can be
fulminant at times.
 In addition to bacteremia these infants may have an
identifiable focus, most often meningitis,in addition to
sepsis.
 Bacteria responsible for LOS and meningitis include those
acquired after birth from human contact or from
contaminated equipment / environment (nosocomial).

8. Late onset sepsis
 The pathogenesis is related to the underlying illness and
debilitation of the infant (esp Preterm, VLBW), the flora in NICU,
degree of asepsis performed during invasive NICU procedures,
breaks in skin and mucosal barrier,etc.
 Organisms most commonly implicated are :
o Coagulase negative staphylocci esp. staph epididermis
o Gram negative rods (eg. Klebsiella, proteus, Pseudomonas)
o Staphylococcus aureus.
o GBS
o Fungal microorganisms.

9. Late onset sepsis
 The pathogenesis is related to the underlying illness and
debilitation of the infant (esp Preterm, VLBW), the flora in NICU,
degree of asepsis performed during invasive NICU procedures,
breaks in skin and mucosal barrier,etc.
 Organisms most commonly implicated are :
o Coagulase negative staphylocci esp. staph epididermis
o Gram negative rods (eg. Klebsiella, proteus, Pseudomonas)
o Staphylococcus aureus.
o GBS
o Fungal microorganisms.

10. Why Microbiology discussion
…???
Information derived from the results of the various
microbiological tests in a NICU setup have an impact on :
Diagnosis of infectious diseases.
Choice of antibiotics.
Antibiotic duration.
Antibiotic policy formulation.
Infection Control measures.

11. Medical microbiology
 Microbes are tiny organisms—
too tiny to see without a
microscope, yet they are
abundant on Earth.
A pathogen or infectious agent
is a biological agent that causes
disease or illness to its host.
These include Bacteria, viruses,
fungi, protozoa.

13. Blood culture
 The isolation of microorganisms from blood is the GOLD STANDARD
used to diagnose sepsis in the newborn infant.
 Despite this fact, Blood culture examination suffers from the
disadvantages of low sensitivity and reporting delay of 24 to 72 h.
 The diagnostic capabilities of blood culture systems have improved over
the last decade with the advent of automated continuous blood culture
monitoring systems.
 Although they can save time, subcultures are required for specific
biochemical or other assays, ultimately needed for pathogen
identification.

14. Sample collection
Technique

15. 1. Prepare the tray for blood culture sample
collection – Gloves, sterile spirit and betadine
swabs, Needle (3), syringe, blood culture bottle.

16. 2. STRICT
Hand Washing.

17. 3. Sterilium &
Gloves

18. 4. Spirit application

19. 4. Betadine application &
Drying

20. 5. Sample
collection

21. 6. Wipe the
blood culture
bottle tip with
spirit
&
Insert blood in
the bottle.

22. 7. Label the bottle correctly &
provide relevant clinical data of
the patient.

23. Blood culture
WHY ????
HOW TO ????
HOW MUCH ???
HOW MANY ???
WHEN ???
HOW ????

24. How much….???
The optimal recovery of bacteria and fungi from blood depends on culturing an
adequate volume of blood.
The optimal volume of blood to be obtained from neonates have not been
defined by certainity, however available data indicates that the yield of
pathogens also increases in direct proportion to the volume of blood
cultured.
Weight of
patient
Patients total
blood volume
(ml)
Recommended volume for
culture (ml)
Total volume for
culture (ml)
% of patients
blood volume
Culture no. 1 Culture no. 2
≤1 kg 60 – 99 2 - 2 4
1.1 - 2 100 - 200 2 2 4 4
2.1 – 12.7 > 200 4 2 6 3Adapted from Kellogg et al. Frequency of low level bacteremia in children from birth to 15 yrs.J. clinical microbiology 2000;38:2181-2185.

25. How many….???
 There is limited information to guide the practitioner on the
optimal number of blood cultures that should be obtained
when evaluating an infant for suspected neonatal sepsis.
 The decreased sampling in neonates is attributed to :
o The small circulating blood volume,
o Potential for increased transfusion requirements,
o Technical difficulties &
o The rapid deterioration of newborns in the setting of
neonatal sepsis.

26. How many….???
Sarkar, bhagat, et al. (2008) studied the usefulness of 2 site
culture in the initial evaluation of neonatal sepsis.
This study strongly indicated that a single site blood culture
with blood volume ≥1ml should be sufficient to document
“true” Gram positive, gram negative or fungal sepsis in
neonates.
An Aerobic bottle should be used for collection of culture of
a neonate.

27. When….???
In contrast with extensive adult data on the periodicity of bacteremia
in a variety of clinical scenarios, neonatal setting is characterised by
a lack of data on timing of blood cultures.
Practically the optimum time to culture for a neonatal suspected
bacteremia is “ As Early As Possible” in the course of septic
episode.
Ideally to be obtained prior to the administration of antimicrobial
therapy.
If already on antibiotics, blood culture to be taken immediately
before administering the next dose.

28. How to read..??
 Interpreting the positive results depends on the clinical
presentation, how the culture was taken, the organisms grown,
and the time taken for the blood culture to become positive.
 Some organisms like N. meningitidis, Candida albicans are nearly
always significant, even in a well looking child.
 Cultures positive with potential pathogens that may also be
contaminants (eg. CoNS) are difficult to interpret and require
clinical correlation for diagnosis.

29. How to read..??
 Rate of contamination are found to be highest in neonates.
 Cultures drawn through indwelling intravenous devices are more
likely to be contaminated and need an additional peripheral
culture sample.
 Positive blood cultures with higher colony counts and flagging
positive within 48 hrs of being drawn have been associated with
an increase likelihood of significance.
 Prior antibiotic use may decrease the sensitivity of the test further.

30. Key Points….!!
Important procedures to improve the sensitivity and
specificity of blood cultures include :
 Proper technique is most critical because it takes only
one organism to contaminate a collection.
 Proper skin disinfection before collection,
 Culturing early in the septic episode.
 Taking an appropriate volume of blood per culture.
 If collecting through an existing intravenous device, ensuring
that a peripheral culture is also collected.
 Wherever practical, more than one bottle per episode. (This
is not always feasible in a very tiny infant.)

32. Fluids sent for culture from NICU :
 Urine
 Cerebrospinal
Fluid
 Pleural fluid

33. Fluids sent for culture from NICU :
 Urine
 Cerebrospinal
Fluid
 Pleural fluid

35. Sample collection
Technique

36. Preparation for
Urine
collection bag
culture
collection
Preparation for
urinary
cathereterised
culture
collection.

37. 2. STRICT
Hand Washing.

38. 3. Sterilium &
Gloves

39. Betadine application
& Drying

40. Catheterised sample collection

41. Urine bag
sample
collection

42. Label the bottle correctly &
provide relevant clinical data of
the patient.

43. Urine culture
Urine culture is essential for
confirmation and appropriate therapy
for urinary tract infection in infants.
(according to a study from the
University of Southern California (USC),
Los Angeles. 2012)
Infants with late onset sepsis tend to
have higher positive results in urine
culture.
In an older infant , a urine sample
collected by aseptic technique (urinary
catheter or suprapubic bladder
aspiration) is an important part of the
sepsis workup.

44. Urine culture
 The frequency of positive urine cultures in infants with early onset sepsis is
relatively low, and it is rare to find bacteriuria in infants with negative blood
culture reports.
 In this era of widespread intrapartum antibiotic use in mother, positive urine
cultures may be obscured because of the excretion of antibiotics in the
urine of the newborn infant.
 It is generally not recommended to obtain urine culture specimens in the
first 72 hrs of life , because the low yield from urine culture.
 Studies have also shown that urine culture collected by bag specimen method
was effective in detecting urinary tract infection only after the 7th day of life.
(falcao mc., Urinary tract infection in full-term newborn infants: value of urine culture by bag specimen
collection.2000)

45. Urine culture interpretation
The colony forming units (CFU) corrosponds to the number of viable bacteria
per ml of urine & is the standard used for the interpretation on urine culture
sample.
A true infection in the absence of prior antibiotic therapy the number of
bacteria is likely to be at least 10000 or more.
Any growth in a sample collected by suprapubic aspiration is considered
significant.
Significant Bacteruria is the numbers of bacteria (≥104
CFU/ml) greater than
those likely to result from contamination from the urethral meatus and its
environs.(ADULT)
Contaminated specimens present with colony counts <10000 and mixed flora.
Gram positive (eg streptococci), fungus and fastidious organisms often are
significant even at lower numbers.
.

46. Key points…!!!
 Prompt plating of the urine sample for culture is important, because
if urine sits at room temperature for more than 60 mins, overgrowth
of minor contaminants can suggest a UTI, when urine may not be
infected.
 Refrigeration is a reliable method of storing the urine until it can be
cultured.
 Multiple (≥3) species of gram negative bacteria - contamination.
Single organism identification with CFU less than 10000 with clinical
significant UTI – warrants treatment.

48. Sample collection
Technique

49. Preparation – Autoclaved gown and sheets, Gloves
(2 pairs), spirit and betadine swabs, needle,
syringe, sterile containers, culture bottle, Bandaid.

50. 2. STRICT
Hand Washing.

51. 3. Sterilium &
Double Gloves

52. Painting & draping …..
Spirit >> Betadine >> Spirit

53. Sample collection Procedure

54. CSF Culture
The incidence of meningitis in neonatal
sepsis varies from 0.3 – 3%.
The clinical features of septicaemia and
meningitis often overlap , thus it is quite
possible to have meningitis with sepsis.
In early onset sepsis, CSF examination
is recommended in presence of a
positive blood culture + signs of sepsis.
In late onset sepsis, lumbar puncture is
advocated in every baby before starting
antibiotics.

55. CSF Culture
 Neonatal meningitis frequently occurs in the absence of bacteremia and in the
presence of normal CSF parameters. No single CSF value can reliably exclude the
presence of meningitis in neonates.
 The CSF culture is critical to establishing the diagnosis of neonatal meningitis.
 Antibiotic treatment prior to lumbar puncture can decrease the sensitivity of culture,
especially when given intravenously or intramuscularly.
 Common Organisms implicated in Neonatal meningitis are : Group B Streptococci,
Escherichia coli, Listeria monocytogenes, Others are Enterobacteriaceae:
Salmonella spp., Citrobacter spp.
 In Infants, these are : Neisseria meningitidis, Haemophilus influenzae,
Streptococcus pneumonia.
• (Garges et al; Neonatal meningitis: what is the correlation among cerebrospinal fluid cultures, blood cultures, and cerebrospinal fluid
parameters. Peds 2006 Apr;117(4):1094-100.)

56. Key points…!!!
• Cultures done on blood agar and chocolate agar remain the gold
standards for diagnosing bacterial meningitis.
• CSF must be processed within 1 hour of collection.
• Do not refrigerate.
• All media should be incubated for 3 days, with daily inspections.
• Blood culture should be done with csf culture. Bacterial meningitis is
often associated with bacteraemia and the causative organism is
sometimes isolated from blood when csf culture is negative.
• Prior treatment is no excuse for not doing an LP!
.

58. ET tube tip
culture
Strict Handwashing
Sterilium + Gloves
Keep sterile scissors and
culture tube ready prior to
extubation.
Tip of the ET tube has to
be cut directly into the
collection tube.
Label the sample
carefully.
Provide clinically
relevant data

59. UVC / PICC line
tip culture
Strict Handwashing
Sterilium + Gloves
Keep sterile scissors and
culture tube ready prior to
extubation.
Tip of the ET tube has to
be cut directly into the
collection tube.
Label the sample
carefully.
Provide clinically
relevant data

60. Swab collections
Strict Handwashing
Sterilium + Gloves
Keep sterile
swab & the
culture tube
ready.
Label the sample carefully.
Provide clinically relevant data
Gently swipe the swab over
the desired site for
discharge collection.
Put the swab in culture tube

61. NICU swabs
• Neonatology is a high-risk specialty for
infection
• Effective infection prevention and
control is central to providing high
quality health care for patients and a
safe working environment for those
that work in healthcare settings.
• Bio-aerosols are airborne particles that
are living (bacteria, viruses and fungi)
or originate from living organisms.

62. NICU swabs
Deepali Danave; Bio-aerosols in Neonatal Intensive Care Units in a District Hospital.
Scholars Journal of Applied Medical Sciences (SJAMS) Sch. J. App. Med. Sci., 2015; 3(5E):2132-2134

63. NICU swabs – surface monitoring
1. Surface monitoring
• Contact surfaces (warmers,incubators) , floors, walls, and other equipment
should be tested on a regular basis.
• Surface Swabs - used for irregular surfaces
Surface monitoring should be performed at conclusion of aseptic processing (to
minimise risk of contaminating critical surfaces during production)
2. Air monitoring
• Settle plates exposed for 30-60 minutes (longer may result in agar drying
out) and replaced for duration of filling.
• Media should be capable of growing a range of bacteria and moulds (e.g.
Soybean Casein Digest Agar (SCDA)/Trypticase Soy Agar (TSA).

64. NICU swabs
3. Water monitoring
• Microbiological quality testing of water is very important.
• Feed water, pre-treatment, purified and water for injection (WFI) should be
tested.
• Water should also be tested for presence of coliforms and/or pseudomonads if
appropriate (may cause biofilm)
• Water used for parenterals should be properly assessed.
4. Compressed Air/Nitrogen/CO2
• Should be periodically tested.
5. Personnel should be periodically tested with surprise audits.

65. BACTEC
An improvement in the
sensitivity and rapidity of
bacterial recovery in blood
cultures has been achieved
with the more recent
automated machines,
including the BACTEC.
Bactec is an automatizated
method to detect the
presence of bacteria in
human blood.

66. BACTEC
The blood is put on a bottle and is
incubated at a certain temperature. The
machine is able to detect the growth of
bacteria in the bottle and alerts the
microbiologist.
The BACTEC cannot identify the
bacteria present in blood, therefore the
microbiologist makes a culture of
the positive bottles.
BACTEC has the advantage that
microbiologist only harvests the
positive samples of blood, making a
quick discard of negative samples with
gaining of time and saving costs.

67. VITEK
• The VITEK® 2 system is a fast,
accurate microbial identification, and
antibiotic susceptibility testing modality.
• The innovative VITEK® 2 microbial
identification system includes an
expanded identification database, the
most automated platform available,
rapid results, improved confidence, with
minimal training time.
• The VITEK® 2 system next-generation
platform provides greater automation
while increasing safety and eliminating
repetitive manual operations.