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Neonatal Sepsis
 Overall incidence = 1-5 per 1000 live births.
 Incidence much higher for VLBW babies :
Early onset seps...
Neonatal Sepsis
 Neonatal sepsis is defined as a clinical syndrome of
bacteremia with systemic signs and symptoms of infe...
Early onset sepsis
 Presents in the first 72 hrs of life and is usually a
multisystem fulminant illness with prominent re...
Early onset sepsis
 Early onset disease is characterized by a sudden onset
and fulminant course that can rapidly progress...
Early onset sepsis
 Early onset disease is characterized by a sudden onset
and fulminant course that can rapidly progress...
Late onset sepsis
 Late onset sepsis is often more insiduous but can be
fulminant at times.
 In addition to bacteremia t...
Late onset sepsis
 The pathogenesis is related to the underlying illness and
debilitation of the infant (esp Preterm, VLB...
Late onset sepsis
 The pathogenesis is related to the underlying illness and
debilitation of the infant (esp Preterm, VLB...
Why Microbiology discussion
…???
Information derived from the results of the various
microbiological tests in a NICU setup...
Medical microbiology
 Microbes are tiny organisms—
too tiny to see without a
microscope, yet they are
abundant on Earth.
...
Blood culture
 The isolation of microorganisms from blood is the GOLD STANDARD
used to diagnose sepsis in the newborn inf...
Sample collection
Technique
1. Prepare the tray for blood culture sample
collection – Gloves, sterile spirit and betadine
swabs, Needle (3), syringe, ...
2. STRICT
Hand Washing.
3. Sterilium &
Gloves
4. Spirit application
4. Betadine application &
Drying
5. Sample
collection
6. Wipe the
blood culture
bottle tip with
spirit
&
Insert blood in
the bottle.
7. Label the bottle correctly &
provide relevant clinical data of
the patient.
Blood culture
WHY ????
HOW TO ????
HOW MUCH ???
HOW MANY ???
WHEN ???
HOW ????
How much….???
The optimal recovery of bacteria and fungi from blood depends on culturing an
adequate volume of blood.
Th...
How many….???
 There is limited information to guide the practitioner on the
optimal number of blood cultures that should...
How many….???
Sarkar, bhagat, et al. (2008) studied the usefulness of 2 site
culture in the initial evaluation of neonata...
When….???
In contrast with extensive adult data on the periodicity of bacteremia
in a variety of clinical scenarios, neon...
How to read..??
 Interpreting the positive results depends on the clinical
presentation, how the culture was taken, the o...
How to read..??
 Rate of contamination are found to be highest in neonates.
 Cultures drawn through indwelling intraveno...
Key Points….!!
Important procedures to improve the sensitivity and
specificity of blood cultures include :
 Proper techni...
Fluids sent for culture from NICU :
 Urine
 Cerebrospinal
Fluid
 Pleural fluid
Fluids sent for culture from NICU :
 Urine
 Cerebrospinal
Fluid
 Pleural fluid
Sample collection
Technique
Preparation for
Urine
collection bag
culture
collection
Preparation for
urinary
cathereterised
culture
collection.
2. STRICT
Hand Washing.
3. Sterilium &
Gloves
Betadine application
& Drying
Catheterised sample collection
Urine bag
sample
collection
Label the bottle correctly &
provide relevant clinical data of
the patient.
Urine culture
Urine culture is essential for
confirmation and appropriate therapy
for urinary tract infection in infants....
Urine culture
 The frequency of positive urine cultures in infants with early onset sepsis is
relatively low, and it is r...
Urine culture interpretation
The colony forming units (CFU) corrosponds to the number of viable bacteria
per ml of urine ...
Key points…!!!
 Prompt plating of the urine sample for culture is important, because
if urine sits at room temperature fo...
Sample collection
Technique
Preparation – Autoclaved gown and sheets, Gloves
(2 pairs), spirit and betadine swabs, needle,
syringe, sterile containers...
2. STRICT
Hand Washing.
3. Sterilium &
Double Gloves
Painting & draping …..
Spirit >> Betadine >> Spirit
Sample collection Procedure
CSF Culture
The incidence of meningitis in neonatal
sepsis varies from 0.3 – 3%.
The clinical features of septicaemia an...
CSF Culture
 Neonatal meningitis frequently occurs in the absence of bacteremia and in the
presence of normal CSF paramet...
Key points…!!!
• Cultures done on blood agar and chocolate agar remain the gold
standards for diagnosing bacterial meningi...
ET tube tip
culture
Strict Handwashing
Sterilium + Gloves
Keep sterile scissors and
culture tube ready prior to
extubation...
UVC / PICC line
tip culture
Strict Handwashing
Sterilium + Gloves
Keep sterile scissors and
culture tube ready prior to
ex...
Swab collections
Strict Handwashing
Sterilium + Gloves
Keep sterile
swab & the
culture tube
ready.
Label the sample carefu...
NICU swabs
• Neonatology is a high-risk specialty for
infection
• Effective infection prevention and
control is central to...
NICU swabs
Deepali Danave; Bio-aerosols in Neonatal Intensive Care Units in a District Hospital.
Scholars Journal of Appli...
NICU swabs – surface monitoring
1. Surface monitoring
• Contact surfaces (warmers,incubators) , floors, walls, and other e...
NICU swabs
3. Water monitoring
• Microbiological quality testing of water is very important.
• Feed water, pre-treatment, ...
BACTEC
An improvement in the
sensitivity and rapidity of
bacterial recovery in blood
cultures has been achieved
with the ...
BACTEC
The blood is put on a bottle and is
incubated at a certain temperature. The
machine is able to detect the growth o...
VITEK
• The VITEK® 2 system is a fast,
accurate microbial identification, and
antibiotic susceptibility testing modality.
...
Microbiological testing in neonatal intensive unit.
Microbiological testing in neonatal intensive unit.
Microbiological testing in neonatal intensive unit.
Microbiological testing in neonatal intensive unit.
Microbiological testing in neonatal intensive unit.
Microbiological testing in neonatal intensive unit.
Microbiological testing in neonatal intensive unit.
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Microbiological testing in neonatal intensive unit.

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Thsi presentation is a sincere attempt to demonstrate the aseptic techniques needed to collect blood culture, urine culture, diagnostic lumbar puncture. Disscussion about the use of there modalities in neonatology practice and the ways to increase their sensitivity and specificity is done.

this presentation is also available in a video lecture format at my Youtube channel - "NeonatoHub". Hope you enjoy it more in that format.
https://www.youtube.com/watch?v=vZ71vymGVC8

Published in: Health & Medicine

Microbiological testing in neonatal intensive unit.

  1. 1. 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)
  2. 2. 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)
  3. 3. 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.
  4. 4. 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.
  5. 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. 6. 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).
  7. 7. 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.
  8. 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. 9. 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.
  10. 10. 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.
  11. 11. 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.
  12. 12. Sample collection Technique
  13. 13. 1. Prepare the tray for blood culture sample collection – Gloves, sterile spirit and betadine swabs, Needle (3), syringe, blood culture bottle.
  14. 14. 2. STRICT Hand Washing.
  15. 15. 3. Sterilium & Gloves
  16. 16. 4. Spirit application
  17. 17. 4. Betadine application & Drying
  18. 18. 5. Sample collection
  19. 19. 6. Wipe the blood culture bottle tip with spirit & Insert blood in the bottle.
  20. 20. 7. Label the bottle correctly & provide relevant clinical data of the patient.
  21. 21. Blood culture WHY ???? HOW TO ???? HOW MUCH ??? HOW MANY ??? WHEN ??? HOW ????
  22. 22. 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.
  23. 23. 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.
  24. 24. 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.
  25. 25. 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.
  26. 26. 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.
  27. 27. 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.
  28. 28. 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.)
  29. 29. Fluids sent for culture from NICU :  Urine  Cerebrospinal Fluid  Pleural fluid
  30. 30. Fluids sent for culture from NICU :  Urine  Cerebrospinal Fluid  Pleural fluid
  31. 31. Sample collection Technique
  32. 32. Preparation for Urine collection bag culture collection Preparation for urinary cathereterised culture collection.
  33. 33. 2. STRICT Hand Washing.
  34. 34. 3. Sterilium & Gloves
  35. 35. Betadine application & Drying
  36. 36. Catheterised sample collection
  37. 37. Urine bag sample collection
  38. 38. Label the bottle correctly & provide relevant clinical data of the patient.
  39. 39. 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.
  40. 40. 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)
  41. 41. 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. .
  42. 42. 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.
  43. 43. Sample collection Technique
  44. 44. Preparation – Autoclaved gown and sheets, Gloves (2 pairs), spirit and betadine swabs, needle, syringe, sterile containers, culture bottle, Bandaid.
  45. 45. 2. STRICT Hand Washing.
  46. 46. 3. Sterilium & Double Gloves
  47. 47. Painting & draping ….. Spirit >> Betadine >> Spirit
  48. 48. Sample collection Procedure
  49. 49. 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.
  50. 50. 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.)
  51. 51. 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! .
  52. 52. 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
  53. 53. 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
  54. 54. 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
  55. 55. 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.
  56. 56. 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
  57. 57. 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).
  58. 58. 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.
  59. 59. 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.
  60. 60. 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.
  61. 61. 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.

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