Sputa Flashcards
What is the main difference between sputum and a BAL?
Sputum = anything the patient is able to cough up themselves
BALS = anything that has to be washed out of the lungs
How is a BAL taken and why do we do them?
- A segment of lung is ‘washed’ with sterile saline after insertion of a flexible bronchoscope, thus allowing recovery of both cellular and non-cellular components of the epithelial surface of the lower respiratory tract.
*Allows for the recovery of both cellular and non-cellular components of the epithelial surface of the lower respiratory tract
What sputa samples are reflex tested for TB?
Why do we do this?
Any sputum labelled as ‘bronchial washes’ i.e. any BALS
This is a screen that we do for TB
A lot of these end up positive
How does an EBUS differ from other sputa samples?
Endobronchial ultrasound-guided transbronchial biopsy
An EBUS will include tissue from a lymph node
How do we grade the appearance of sputa
○ Salivary
○ Mucosalivary
○ Mucoid
○ Mucopurulent
○ Purulent
○ Blood stained
○ Bloodstained and mucopurulent
○ Bloodstained and purulent
What samples do we describe the appearance of?
Sputa only
Describe the processing of sputa
Book in sample -> assign a lab number
Describe appearance
If any out of house test label up an ependorf as needed and aliquot sample
Add equal parts sputasol and vortex
Leave for 20 mins
Add 10ul of sample to each plate, for dilution plate add the 10ul to 20ml sterile distilled water and then inoculate plate
Streak plates using separate loops
Add a C30 disc to chocolate plates
Add optochin to blood agar
For how long should a sputa sample be left in sputasol
20 minutes
What does sputasol do
○ Breaks down the sputa to release any bacteria which might be there
○ Makes the sample a lot easier to work with -> easier to spread on plates etc
How much of a sputa sample is used to inoculate plates?
10ul (of equal parts sputasol and sample)
What plates are sputa samples put up on?
Blood agar
Chocolate
Chocolate dilution plate
1/4 of a Candida chromagar plate
How do you make up a chocolate dilution plate?
○ 10ul of sample in 20ml of sterile distilled water (mix by vortex)
○ 10ul of this diluted sample is then used to inoculate the chocolate agar
What disk is put up on the blood agar for sputa samples and why?
Optochin
For S. pneumo ID
What disc is put up on chocolate plates for sputa samples and why?
C30/Chloramphenicol
What are some add on/supplementary tests for BALS
Galactamannan
Fungal PCR
PCP (pneumocystis pneumoniae)
Aspergillus lateral flow
How are BALS processed?
Book in -> give lab number
Aliquot for supplementary tests
Mucoid BALs need to be mucolysed for 20 mins
All BALS centrifuged
Pellet is sent to TB
Majority of supernatant is decanted off
Resuspend remaining liquid/tissue
Inoculate plates and slope with 1 drop
C30 on chocolate
Optochin on blood
What BALS are centrifuged?
ALL BALS other than those of known Covid19 positive patienets
What are BALS put up on
Blood
Chocolate
MacConkey
PDA slope
Candida plate
What is put up on chocolate plates for BALS
C30
What is put up on blood for BALS
Optochin
Why is there no chocolate dilution plate for BALS
▪ No need for quantification as we pick anything that grows for BALS
▪ If anything has grown on the chocolate we are picking it so no need for the dilution plate
How long are PDA slopes for BALS incubated
get 5 days incubation but checked regularly
What is our control for our PDA slopes?
open slope and wave around
What plates do EBUSs get
Same as BALS but also get an FAA with a mt disc
What extra plates do transplant patients get
(Same as BALS +)
CxSA
MRSA
FAA + Mtz
B. Cepacia
Own Candida plate
What supplemental tests do BALS for post transplant patients also get?
Lateral flow for aspergillus
Who gets a Burkholderia plate and why
○ Done for ICU/HDU patients
○ Done for CF patients
○ Any transplant patient under aged 55
○ Done as a screen for any patients entering ICU
Outbreak last year
No positives since initial positive during outbreak
How are BC plates read
Read on day 2 with other plates -> interim report
Put up for an additional 3 days and read again
What does a Burkholderia positive mean?
○ A positiev Burkholderia would mean removal from the transplant list for a patient
▪ Burkholderia causes necrotising pneumoniae
Failure of transplant etc
What will grow on BC plate
B. Cepacia
Some GNBs such as seratia sometimes grow
What extra plates do CF patients get?
Same as BALS
+ SA
+ MRSA
How are sputa plates read
Read twice, first on day 1, second on day 2
How are the colony counts calculated using the chocolate dilution plate
○ 3 or < colonies = 10^6
○ 3 – 25 colonies = 10^67
○ 25+ colonies = 10^7
What should you do if more than 25 colonies on a plate?
> 25 colonies on plate should be ID’d, might just come back as a commensal but better to be safe than sorry
Write about S. aureus in sputa
▪ Clox/C30 sensitive
▪ Usually display a black halo on chocolate agar
▪ On day 2 will appear orangey on MacConkey agar
▪ If query staph carry out a staphyslide agglutination test to confirm
○ If positive p0ut up a purity plate for sens (don’t have to ID/MALDI first unlike with GNBs)
▪ Gram + clusters, grape like clusters in 2, 4, 8, etc
▪ If query SA then staphaurex card agglutination test
* SA = SS+
* SS- colonies do not have to be picked for ID (unles requested)
▪ DNase agar +
▪ Tube coag +
▪ API biochemical tests or VITEK can also be used to ID SA
▪ Should grow as golden colonies on chocolate but are most of the time a pale yellow/white
▪ On chocolate SA will often grow with a dark halo around the colonies
▪ Will appear orange coloured on MacConkey after 2 days growth -> may even be coral coloured but are still a NLF ?
SA should always be picked if sen
What are the most common community acquired organisms seen in sputa?
Why is this important?
H. influenzae, S. pneumoniae, M. catarrhalis, S. aureus, MRSA, Pseudomonas
If we query any of these they should always be picked and ID’d
Write about S. pneumo in sputa
▪ If query can put up a purity plate with an optochin disc
* Optochin between 1’ and 2’ inoculum
* Hoping for pure growth on PP
▪ MALDI cannot ID S. pnemo well which is why optochin susceptibility is so important
▪ Alpha haemolysis (green haemolysis)
▪ Optochin disc suscpetible i.e. zone of clearance
▪ If query S. pneumo -> put up a purity plate to rule out pseudoneumo
Optochin disc in the centre
Write about H. influenzae in sputa
▪ Grows as grey coloured colonies on chocolate agar
▪ Small, round, grey colonies
▪ Will grow right up to C30 disc on chocolate
▪ Tell tale sign is growth only of colonies on the chocolate agar plate only
* X and V factors only present in chocolate
Gram negative coccobacilli
Write about M. Caterallis in sputa
▪ Gram negative diplococci
▪ Catarrhalis = oxidase and catalase positive
Hockeypuck colonies
Catalase positive
Write about Pseudomonas in sputa
▪ Non Lactose fermenter (yellow colonies on MacConkey)
▪ Oxidase positive
▪ Usually dark colonies on Chocolate agar
▪ Pseudomonas aeruginosa will grow greenish – may have a metallic sheen to it
▪ Pseudomonas aeruginosa is the most common infectious agent in CF and COPD or any intubated patients
Resistance is common
Write about BHS in sputa
▪ Only of interest in bronchial washings/BALS etc
▪ Part of normal throat flora so not of interest in normal sputa
▪ i.e. BHS only significant if not part of expected flora
But if pure growth of a BHS then you should pick this as it is may be the infecitous agent
Write about GNBs in sputa
▪ If growth = 6 we don’t pick, if from a normal ward/GP
▪ Colony count greaer than 25 needed for a GNB to be picked from a GP sample i.e. growth of 10^7 cfu/ml on chocolate dilution plate
▪ GNBs are rarely infectious in healthy patients so there needs to be a large number of them in order to suspect infection
▪ We only pick a GNB for our immunocompromised patients or if there is a lot of growth
▪ These tend to be commensals and rarely cause infection in healthy population
▪ Tend to be big wet and gloopy colonies on the MacConkey (majority that you will see are non-lactose fermenters)
▪ We don’t pick GNBs for normal patients if colony count of 6
▪ Lactose fermenters= E. Coli, Klebsiella, Enterococci
Non lactose fermenters = Pseudomonas, proteus
Write about yeasts/fungi in sputa
▪ All fungi are very important in our immunocompromised patients e.g. our ICU or transplant patients
* Hence why these patients get their own candida plate instead of the shared one between 4 for notmal sputa
Want to be super confident that any fungi on the plate is actually infectious and not just contaminants
▪ We type any fungi for our transplant patients -> need to know if candida or not
▪ Candida and Aspergillus are the most commonly seen infectious fungi
▪ Only have to ID C. albicans for immunocompromised patients e.g. ICU, HDU, transplants etc
Fungi cause fungal pneumoniae e.g. pneumocystis jirovecci which causes life threatening pneumoniae etc
How do we ensure any query fungi are infectious and not contaminants
Put up done in cat 3 => aseptic
○ Normally we have the same person doing the put up that is doing the reading to ensure confidence in aseptic technique etc
MS should be confident in their technique in that any fungi found are trtuly infectious etc
How do we ID our fungi
▪ Fungi can be ID’d using the MALDI or through microscopy by a specialist scientist
* Light microscopy ID
○ Tape technique + blue dye (?)
○ Looking for features e.g. phylites etc
○ Tape technique can be done on day one -> if query fungi -> great if urgent and cant wait for MALDI ID
If any hyphae/apores are seen you can put up a PDA plate to be read on day 2
For what patients do we have to ID any fungi
For any immunocompromised patients e.g. ICU/HDU/transplants etc
Only have to ID C. albicans
What safety considerations should be taken for when working with fungi
- When working with aspergillus be very careful to not inhale any of the spores
* Any MS with asthma should wear a mask when working with aspergillus
* Wrap all PDA plates in parafilm to prevent contamination of plates as well as accidental inhalation of fungal spores
Nobody should be inhaling aspergillus spores ever
Talk about sensitivities in fungi
- Resistance against Azoles
* VIPs
* Fungi e.g. Aspergillus will go for VIP sensitivity testing for anti-fungal treatment
* Looking for resistance against azols
* Controls: positive and negative resistance against azoles
○ Each get own VIP plate
* For every VIP we put up a pos and neg control
○ One organism is sensitive to the antifungal in each well
○ Resistant control is resistant to the antifungals in each well
* Also sent out to Bristol for further sensitivities
What are the most commonly found commensals in sputa
Streps and Neisseria species
What organisms do we always pick for BALs and why?
We pick anything that grows as BALS should be sterile
What should you do if there are multiple organisms on a plate
- If multiple organisms put up a purity plate for each colony type
○ Avoid mixed purity plates as this greatly delays sensitivites
What infections are common in CF patients
- Infections usually start of with Staph infections
- Infection develops into strep infection e.g. S. pneumo
- As fluid builds up in the lung organisms such as Pseudomonas begin to take over as these prefer wet conditions
- Prolonged antibiotic use in these patients leads to resistant Pseudomonas strains
○ There is a new drug available for these patients which means they arent havening to undergo lung transplants
▪ We don’t know how long this antibiotic will work for though before resistance occurs again - Patients in end stage of the disease tend to get more serious infections such as Burkholderia
○ This means removal from transplant list
▪ Can be a death sentence for these patients
Burkholderia -> necrotising pneumonia -> transplant rejection
How long does it take most purity plates to grow
Most grow in only about 5 hours
Query streps are often put up anaerobically overnight though
Do we do gram stains for sputa/BALS etc
Gram stains only done on request
Grams only really made for new lung transplant patients
Why do we do a dilution plate/colony counts
- Done mostly for GP samples using dilution plates
- Qualitative measurement of infection
A higher colony count is needed in GP samples in order to query a GNB as an infectious agent
- Qualitative measurement of infection
How long can a sample be left in sputasol
Sample should be processed immediately but organisms should survive for approximately 48 hours
