TB

Question 1

What are the two types of TB we see in the lab?

Answer 1

Mycobacterium TB
Non mycobacterium TB

Question 2

How many people have latent TB?

Answer 2

1 in 4 Irish People

Question 3

Where is TB most common?

Answer 3

Seen in people travelling from poorer, overcrowded countries e.g. India

Question 4

How is TB treated?

Answer 4

6 months antibiotic cocktail treatment containing rivampicin

Question 5

What kind of TB investigations do we get from the IBTS?

Answer 5

• We also get TB investigations from the IBTS
  Preservative fluid or donation tissue

Question 6

Why do we get so many repeat TB samples

Answer 6

○ Docs need three consecutive negatives to say a patient is negative
▪ Hence the overload of samples
Docs will even go off three negative consecutive auramines

* We are looking to limit the amount of samples can be sent for a positive TB patient  We want a limit of 3 samples a month

Question 7

For how long do we keep samples?

Answer 7

At least one month

Question 8

What sample types do we see for TB

Answer 8

• TB testing is done mostly on sputa and BALs
  
  • Urines
  • CSFs
  • Tissues
  • Fluids e.g. pleural fluid
  • IBTS material
    ○ Preservative fluid from organ donation
    Donation tissue itself

Question 9

Describe how TB samples are processed

Answer 9

• Any sputa samples or BALs must be put in sputasol for 20mins
  
  • Samples must be spun for 20 minutes @ 3000 rpm
  • Any tissue smust be mushed up and suspended in distilled water and then spun -> as with sputa/BALs
• Auramine slide is made
  
  • After slide is made, spin down sample again and pour off the supernatent
  • 1.5ml TB buffer solution is added to all pellets
  • 0.5ml to TB bottles and put on BacT for 7 weeks
  • Takes 9-15 days for TB to grow

Question 10

Why do we mush up our tissue samples?

Answer 10

The softer and mushier the fluid the better, especially for the IBTS organ donation screen

Question 11

How are auramine slides made

Answer 11

○ Auramine slides are made using a diamond topped pen
○ Slides are heat fixed @ 70 degrees for 1 hour
Decontaminate with NaOH (sputa, urine + BALs) for exactly 20 minutes
- Manually stained
-

Question 12

How is NAOH used

Answer 12

○ Decontaminate slides with NaOH (sputa, urine + BALs) for exactly 20 minutes
▪ All other bacteria will not survive the 20 minutes but TB will survive
▪ NaOH is then neutralised after 20 minutes
* Neutralised with buffer -> colour change from fuchsia to salmon orange

Question 13

How long are TB blood cultures left onboard the BacT?

Answer 13

7 weeks

Question 14

How long does it take a positive TB to grow on BacT?

Answer 14

9-15 days

Question 15

What is done with any positive BacTs

Answer 15

Any positives are taken off and an auramine slide is made

Question 16

What would you do if the auramine slide is positive but there is no growth?

Answer 16

Send for GeneXpert
-> any atypical TBs will come up as GeneXpert negative

Question 17

Where do we send typical TBs?

Answer 17

IMRL reference lab (Irish Mycobacterium Reference Lab in St. James’)

Question 18

Where do we send any atypical (GeneXpert negs) to

Answer 18

Scotland reference lab

Question 19

What happens to all BacT bottles on say 42?

Answer 19

All bottles are taken off after 7 weeks

A terminal auramine is made regardless if positive or negative

Question 20

What samples can be used on the GeneXpert

Answer 20

Only sputa and BALS are suitable

Question 21

Other than Typical vs Atypical TB what can the geneXpert tell us?

Answer 21

If the TB is rivampacin resistant or not

-> confers a much more severe form of the disease

Question 22

How do you prep a sample for GeneXpert

Answer 22

○ 600ul of sample in 1.5ml buffer incubated for 15 minutes
○ Vortex sample, leave, vortex again
Avoid bubbles

Question 23

What are the three auramine slides made?

Answer 23

Initial auramine -> nearly always negative
BacT positive auramine
Terminal auramine

Question 24

For what samples do we not do an auramine and why

Answer 24

Urines or pleural fluids dont get an auramine

We just never see the bacilli in these samples

Question 25

How do you read the auramines

Answer 25

○ Bacilli will fluoresce green
○ Bacilli tend to be beaded
Don’t confuse with GNBs
○ Non specific green staining is seen in most negatives
Be careful not to read the slide too close to the edge as glass shards will fluoresce

Question 26

What should you do if you query GNB on an auramine

Answer 26

Can always do a HAIN stain

Question 27

How will atypical TB look on the auramine?

Answer 27

▪ Tend to be short and stumpy
Will still be very fluorescent

Question 28

How do you record contaminants on an auramine?

Answer 28

▪ Gram positive (small)
▪ Gram negative (large, juicy bacilli)
Important to record these but anything other than TB doesn’t mater

Question 29

How do we control our TBs?

Answer 29

Positive control for all auramine slides

Question 30

Why do we send atypicals out

Answer 30

Sent out to type the atypicals (upon request by doctors)

Question 31

What is MGIT

Answer 31

Mycobacteria Growth Indicator Tube
Consists of liquid broth medium that is known to yield better recovery and faster growth of mycobacteria
Contains 7.0 ml of modified Middlebrook 7H9 broth base

Atypicals will still be negative even after use of broth

Question 32

What do we do with positive TBS in general

Answer 32

They are cultured onto Middlebrook broth base and frozen

They are stored indefinitely

Question 33

How does one remain safe in Cat3

Answer 33

Ethanol spray + AZO wipes are used to decontaminate surfaces

Chemgene will not work for TB

All waste is autoclaved before leaving Cat3