Journey of a TB Specimen

1
Journey of a TB Specimen

• Andrea Warman, FNIHB
• Evelina Der, AHS

2
specimen collection
specimen processing
smear


PCR
culture




report as TB complex
hold for 7 weeks
probe


report as negative
report as TB
microbiology identification

drug susceptibility testing bacterial typing
3
Workflow

• Community
• specimen collection
• specimen transportation
• Provincial Laboratory
• specimen processing
• isolation identification
• susceptibility testing
• bacterial typing
• Reporting
• Provincial Laboratory ? TB Services/Clinics/Orderi
  ng Physician ? Public Health Nurse

4
When to collect sputum

• When someone presents with symptoms of active
  disease
• When you are making a referral to TB Services
• When someone has been a contact of an active TB
  case
• Note Trust your own instinct, it is never wrong
  to collect sputum

5
Tips for collecting sputum

• DO NOT rinse mouth prior to collection
• 3 early morning specimens best one anytime
  better than none
• Ensure lid is tightly secured (no leakage to
  avoid recollection)
• Keep in fridge or cool place to avoid overgrown
  specimens (? recollection)
• Ideal specimen is more than 2 ml (enough to cover
  bottom of jar)

6
Requisition Form

• Fill in the following fields
• Client name
• PHN
• Birthdate
• Date of specimen collection
• Ordering physician/agency
• Specimen Type (sputum)
• Test Requested (AFB)
• Copy to

7
Specimen transportation

• Transport specimen as per regional guidelines
• Make sure lid of specimen jar is closed correctly
• Ensure specimen jar labeled with pt name, DOB,
  PHN, collection date
• Place into secondary watertight container with
  absorbent material
• Completed requisition into pocket
• Place into rigid outer packaging
• Protect from temperature extremes

8
Specimen Types

• Respiratory
• sputum (including induced)
• induced sputum similar in quality to bronchial
  specimens
• in children, specimen of choice is gastric
  aspirate
• bronchial wash (bronchial alveolar lavage BAL)
• Tissue or aspirate
• lung
• pleural
• lymph node, etc
• Others as applicable

9
Possible Problems with Specimens

• contamination from flora
• to avoid overgrowth, refrigerate specimen after
  collection and transport timely
• contamination from environmental AFB
• to avoid contamination, dont expose to tap
  water (e.g. rinse mouth with water prior to
  sputum collection), etc.
• limited use of swabs
• if swab is the only choice, submerge gauze in
  sterile saline solution
• insufficient quantities of specimen
• for respiratory, needs about a teaspoon full

10
Preparation of specimen
specimen
digestion-decontamination
centrifuge
inoculation for culture media
microscopy
Auramine-rhodamine screen
Ziehl-Neelson confirmation
solid media
liquid media
11
Identification by smear (1)

• Ziehl-Neelsen (ZN) stain
• Stain with carbol fuchsin (a mixture of phenol
  and basic fuchsin), decolourize with
  acid-alcohol, and counterstain with methylene
  blue
• Mycobacterium will appear as pink, on a blue
  background

12
Identification by smear (2)

• Auramine-rhodamine (AR) stain
• Stain with specific fluorescent dyes, and
  visualize using fluorescence microscope 
• Mycobacterium will appear as reddish-yellow
  fluorescence

13
Identification by Polymerase Chain Reaction (PCR)

• Only done on smear positive respiratory specimens
• Amplification and detection of a specific RNA
  sequence in M. tuberculosis
• to confirm M. tuberculosis complex
• not done on smear positive rare specimens
• sensitivity 90-100, specificity 92-100

14
Identification by culture

• All specimens are processed using 2 separate
  medium (liquid and solid) except specimens of
  bone marrow and blood
• Quicker result by liquid medium
• Confirmatory result by solid medium

15
Identification culture on liquid media

• The BACTEC system (Becton-Dickinson Microbiology
  Systems) is an automated liquid media system for
  rapid detection of mycobacterial growth
• Isolation of M. tuberculosis in Mycobacterium
  Growth Indicator Tube (MGIT) system has faster a
  faster turnaround time than solid media
• Smear positive respiratory fluid within 10 days
• Smear positive tissue up to 3 weeks
• Smear negative samples 10 28 days

16
Identification culture on solid media

• LowensteinJensen (LJ) medium, is an egg-based
  solid medium for culture of mycobacteriumagar-base
  d
• Middlebrook medium is an solid medium for culture
  of mycobacterium
•  tends to grow fewer contaminants than the
  egg-based media

17
Confirmation by DNA probe

• a fragment of DNA or RNA of variable length is
  used for detection of the nucleotide sequences
  (the DNA target) presence to identify the
  mycobacterium
• DNA probe is done on culture positive specimens
• Positive M. tuberculosis complex
• Negative Nontuberculous mycobacteria (NTM) such
  as M. Avium Complex (MAC), M. kansasii, M.
  gordonae

18
Drug Susceptibility Testing

• to determine effectiveness in stopping the growth
  of mycobacterium
• each drug is tested individually to confirm
  effect on the specimen
• to establish proper drug treatment regime
• if drug resistance is noted, need to modify
  standard treatment regime
• results are generally ready in 10 days

19
Bacterial Typing

• DNA fingerprinting by Restriction Fragment Length
  Polymorphism(RFLP)
• different isolates of M. tuberculosis will have
  different IS 6110 fingerprint patterns
• these can be compared to
• determine clonal identity within
• a cluster
• provide insights into
• contact tracing

20
specimen collection
specimen processing
smear


PCR
culture




report as TB complex
hold for 7 weeks
probe


report as negative
report as TB
microbiology identification

drug susceptibility testing bacterial typing
21
Reporting What does it mean? What does it mean
to your practice?

• Smear
• Positive vs Negative
• Number of s
• PCR
• Culture
• Positive vs Negative
• Probe
• Sensitivities
• Fingerprinting