Tuberculosis - Laboratory Investigation Flashcards
What are the four levels to laboratory diagnosis of MTB?
Smear/Direct microscopy
Rapid molecular diagnostic tests
Culture media both solid and liquid
Drug susceptibility testing (molecular assays, liquid or solid medium methods)
What are some of the benefits/downfalls of each diagnostic method for MTB?
Microscopy -> same day detection but poor sensitivity
Line probe assays -> first 24/48 hours
Direct culture and susceptiblity takes weeks to months
What is suggested as the best method of ast for tb and why?
Reference labs suggest direct susceptibility as the gold standard
Molecular methods can only detect the presence of genes known to confer resistance but this doesnt always represent the clinical picture
Molecular methods are also unable to identify resistance due to new mechanisms/genes -> if the mutation is new or unkown then it will come up falsely negative
Give a quick breakdown of the lab investigation for tb
specimen receipt into ab within 24 hrs of specimen collection
process and concentrate sample (decontaminate)
Acid fast microscopy -> report <24hrs
Liquid culture e.g. automated MGIT
Recovery of organism (10-14 days)
Identification of species (21 days of recepit)
Susceptibility testing
Talk about decontamination step for TB
NACL NaOH used to decontaminate specimen of any respiratory tract flora/oral flora
Talk about decontamination step for TB
NACL NaOH used to decontaminate specimen of any respiratory tract flora/oral flora
From a lab point of view how do we confirm a TB
Isolation of MTC (excluding M. bovis BCG) from a clinical specimen e.g. liquid culture positive
OR
Detection of MTC nucleic acid in a clinical specimen
AND
positive microscopy for AFB
i.e. positive liquid culture or NAAT as well as AFB on smear
What kind of specimens are suitable for TB investigation?
Non Sterile Sites such as:
- pulmonary: sputa, BAL, BRW, BA
- renal: urine
Sterile sites:
- pleural fluids, joint fluids, CSF etc
- tissue
What kind of specimens are suitable for TB investigation?
Non Sterile Sites such as:
- pulmonary: sputa, BAL, BRW, BA
- renal: urine
Sterile sites:
- pleural fluids, joint fluids, CSF etc
- tissue
What kind of sample is required for query TB meningitis?
2 CSF samples needed to enhance senstivity
Give some exampls of inappropriate specimens for TB investigation
Faeces -> commensal mycobacteria, difficult to interpret, difficult to decontaminate
Urine for the investigation of pulmonary TB
How should sputa or urine be collected?
clean, sterile plastic container
Before commencement of therapy
Early morning samples
Procured on 3 consecutive days
5-10mls ideally but min 2mls
Non salivary sputa
Not pooled
Refrigerated if delay in transport
Why is an early morning sample ideal?
Lying down all night -> aids pooling of bacteria -> first expectorate/urine ideal -> collection of bacteria
Highest bacterial count
What culture media is used for TB?
MGIT -> liquid culture
Lowenstein-Jensen (LJ) slopes - solid
If AFB negative smear what should you do?
Usually wait for consultant to request a molecular test as AFB neg doesnt always mean TB neg
High clinical suspicion important
How do we carry out molecular detection of TB?
Direct detection of nucleic acid using the GeneXpert
What are some of the main safety considerations surrounding TB specimen processing safety?
(7)
Environmental persistence of TB - think of resistant cell wall
Aerosol formation + inhalation
Hazard Group 3 pathogen
Cat3 lab + biosafety cabinet
Centrifugation
Disinfection and autoclaving of equipment
Vaccination of staff and mantoux testing if necessary
What kind of lab is TB processing done in?
(3)
Must be done in a category 3 containment lab
Must be done in a biosafety cabinet with an installed air filtration system with HEPA filters insalled
How persistent is TB in the environment?
TB can resist disinfection and somatic stress
What risk is there surrounding centrifugation of TB specimens?
Poses the risk of aerosols and therefore inhalation
Risk of breakages
Sealed buckets must be used to prevent aerosols
How is equipment disinfected post TB processing?
Disinfected in hypochlorite solution and then autoclaved
How should staff be prepared for TB processing?
Staff should be appropriatly trained
BCG vaccinated
If exposed or signs of symptoms etc then Mantoux skin test should be carried out
Why do we need to decontaminate sputa for TB testing?
There are lots of commensals present in sputa samples -> these will overgrow/outgrow any mycobacteria if allowed to do so
We want to kill as many of these commensals while preserving as much mycobacteria as we can
What is the most common method of decontamination for TB samples?
2% NaOH
This oftens involves a mucolytic agent such as N-acetyl-L-cysteine (NALC) or sputasol
What is the ideal contamination rate of a sputa sample?
Between 2-5% required
<2% is too harsh and you will kill too much mycobacteria
>5% is too weak and the mycobacteria will become overgrown
How do we decon for TB?
NaCL NaOH made up daily
Equal volumes used
Vortex for 20 seconds
Let stand at room temperature for 15 mins
Does decon interfere with downstream culturing or detection methods?
NO decon using NaCl NaOH is validated on both the MGIT and the LPAs
What does MGIT stand for?
Mycobacterial Growth Indicaor Tube (MGIT)
Talk about the use of microscopy for TB
(3)
Microscopy should be performed after homogenisation but before decontamination, or it should be done directly from samples
Smear positives indicates presumptive diagnosis of TB and infectivity i.e. if smear positive then def positive and infectious
Smear negative doesnt necessariy mean negative but indicates less infectious
Talk about the use of microscopy for TB
(3)
Microscopy should be performed after homogenisation but before decontamination, or it should be done directly from samples
Smear positives indicates presumptive diagnosis of TB and infectivity i.e. if smear positive then def positive and infectious
Smear negative doesnt necessariy mean negative but indicates less infectious
What are the five main benefits of carrying out TB microscopy?
Used to determine whether isolation is required -> are they infectious or not
Influences the extent of contact tracing - more tracing needed if highly infecitous etc
Used to monitor treatment - negative smear good indicator that treatment is working
Results should be available within one working day
Both viable and non-viable organisms will stain
What are the two smear stains available for TB?
Auramine-O stain
Ziehl Neelson
Compare the use of Auramine O versus ZN?
Limit of detection about 10^4 bacilli/ml (10,000) for AFB but 5x10^3 (5000) for ZN i.e. need more bacteria for AO
However you dont need a fluorescent microscope for the ZN stain which makes it a better choice for high burden countries such as Africa
Compare the use of Auramine O versus ZN?
Limit of detection about 10^4 bacilli/ml (10,000) for AFB but 5x10^3 (5000) for ZN i.e. need more bacteria for AO
However you dont need a fluorescent microscope for the ZN stain which makes it a better choice for high burden countries such as Africa
What are five factors that affect the sensitivity of smears?
Quality of specimen
If prepared prior to or post decontamination
Centrifugal force
Type of stain (auramine/direct Zn)
Experience of Reader
What is the requirement for a positive ZN?
Magnification at 1000 or >oil for 15 minutes or 300 fields
-> must see 1 bacillus
What is required for a positive Auramine O?
Magnification of approximatel 150 dry (not oil) for 2-3 minutes 30 fields (80)
Must see 3 bacilli
In routine labs why is auramine O the preferred stain for TB?
(2)
10% more sensitive than ZN with similar specificity
Lower magnification and less time required
what % of culture positive specimens are smear positive?
About 50% of culture positives are smear positive
What are five limitations of microscopy for TB?
Cannot distinguish between dead and living bacilli
High bacterial load >3000-5000AFB/ml is required for detection
Cannot distinguish between species
No indication of drug susceptibility
Some patient cohorts will have a much lower amount of TB in sputa -> HIV and children will always have a lower bacilli count
What is the go to solid media for TB?
Lowenstein-Jensen medium slopes
What does the LJ slopes contain?
Eggs
Malachite green
Glycerol (for MTB) or pyruvate (M. bovis)
PANTA cocktail
What are the components of the PANTA cocktail?
Polymyxin
Amphotericin
Nalidixic acid
Trimethoprim
Azlocillin
What does the malachite green component of LJ medium do?
Malachite green -> mycobacterium quite resistant to this but everything else is susceptible
What must be added to Lowenstein-Jensen media to favour growth of MTB vs M. bovis
For MTB add glycerol
For M. bovid add pyruvate
What are the pros and cons of LJ slopes?
- 4 of each
Can detect as few as 10 viable cells
Easy and economical to prepare
Lower contamination rates then with liquid media
Isolated colonies with characterisitic tough, rough and buff colonial morphology can be observed
MTB takes between 14-28 days to grow but can take 8 weeks to grow if patient is on treatment
Need to be checked at regular intervals
If contamination does occur it covers the total surface of the medium
DST difficult to perform using egg-based media
Why is drug susceptibility testing more difficult to perform using egg-based media?
This is because some drugs must be adjusted to account for their loss by heating or by interaction with certain components of the egg
What is the go-to liquid media for TB?
(2)
Middlebrook 7H9 Broth
Its supplemented with glycerol to make it more suitable for M TB growth
What are the pros and cons of liquid media?
Growth is more rapid in liquid media vs solid
Contamination and mixed cultures are more common and difficult to interpret
What is the go to method of automated mycobacterial culture in most labs?
Mycobacterial Growth Index Tube (MGIT) system
Write a note on the MGIT.
- How does it work?
- How many and what samples?
(6)
Its a rapid liquid culture method
Utilises fluorescence technology triggered by O2 reduction
O2 continuously monitored over a 6 week protocol
Automatically reads cultures
Capable of holding 960 patient samples
Can be used for the majority of specimens in the TB lab
How long does it usually take for a positive on the MGIT?
(2)
Usualy takes 10-12 days for a MGIT positive
The MGIT protocol is 6 weeks though so samples will stay onboard and be continuously monitored until then
What kind of medium is used onboard the MGIT?
Middlebrook 7H9 broth containing OADC and PANTA Cocktail
What technology is used onboard the MGIT?
Fluorometric technology
What is the principle behind fluorometric measurement on the MGIT?
A fluorescent oxygen sensor is embedded in the base of each tube
This sensor detects any decrease in O2 dissolved in the broth
The oxygen sensor will emit light when exposed to UV
Actively respiring organisms consume O2, decreasing concentration
This reduction is detected by the MGIT
MGIT then flags tube as positive for scientist
After a positive liqud culture what is the recommended next step?
To confirm with a ZN stain
-> centrifuge tube for 15 mins and stain using ZN
-> note appearance of mycobacteria (cording or non-cording/clumping)
What does cording TB in a flagged MGIT tube indicate?
Typical of Mycobacterium TB
What does non-cording/clumping bacteria in a ZN of a positive MGIT tube indicate?
Non-TB-Mycobacterium species
What are the three limitations of culturing TB?
Turn around time (up to 8 weeks for solid and 6 for liquid)
Training -> staff need a lot of training
Cat 3 facilities are required
When should susceptibility testing be done
When the patient is first found to have positive culture for TB
Ideally before treatment
Write a note on Susceptibility testing for TB, where and why do we do it?
Only done in the referene lab
Really important to determine what is the best regime for the patient -> think long antibiotic course so want to get it right the first time
Phenotypic methods arent done as these would takes weeks ontop of the weeks/months for culture positives
What methods are there of susceptibility testing for TB, what is the preferred method?
Solid media on LJ medium but this takes 3-4 weeks
Liquid media on the BACTEC or MGIT but this takes 7-10 days
Molecular methods - takes only a few hours - 1 day
Phenotypic DST remains Gold standard as molecular can miss mutations
How is phenotypic ST carried out on the MGIT?
(3)
Multiple tubes used -> one growth control and one tube for each drug tested
A known concentration of each drug is added to a MGIT tube along with the specimen -> the growth of specimen is compared to the drug-free growth control -> same amount of specimen added to each tube aswell
If the drug is active growth will be inhibited and fluorescence will be suppressed while the drug free control will show increasing fluorescence
If the isolate is resistance growth and fluorescence will be commparable to that of the control
Talk about broth microdilutions as a form of ST for TB?
(4)
A new method of ST
Trek Sensitre plates
Increasingly being used
Results were often interprete against EUCAST breakpoint established for other methods e.g. agar or MGIT but in 2020 EUCAST provided new guidelines for these as an MIC process
What is the GeneXpert MTB/RIF Assay, what does it do?
(3)
Automated NAAT, Real time PCR for detection of TB and Rifampicin resistance detection
Endorced by WHO in December 2010
11 chamber self-contained PCR
What are the main benefits of the GeneXpert MTB/RIF assay?
(7)
A closed system with minimal hands-on technical time
Provides rapid results in less than 2 hours
Does not require a Cat 3 lab -> good for developing countries
Sample preparation, amplification and detection all integrated on board
Dry interfaces - minimal contamination risk
Precise assay - computer controlled process and pipetting - less room for error
Quality assured - extraction controls, probe-check controls and each cartridge has its own PCR control
What are the steps to using the MTB/RIF test on the GeneXpert
(8)
Sputum liquefaction and inactivation with 2:1 sample reagent -> incubated for 15 mins
Transfer of 2ml material into test cartridge
Insert onto test platform
Sample automatically filtered and washed
Ultrasonic lysis of filter-captured organisms to release DNA -> this chemically inactivates the bacteria
DNA molecules mixed with dry PCR reagents
Seminested rtpcr amplification and detection in integrated reaction tube
Printable test result
What are the targets for MTB and RIF on the GeneXpert
rpoB gene of M. tuberculosis complex
Talk about the use of the rpoB gene to detect TB and Rif on the GeneXpert
(6)
GeneXpert amplifies u a sequence of the rpoB gene where 95% of genes responsible for Rifampicin resistance are found
The GeneXpert utilises 3 specific primers and 5 unique molecular probes
Together the 5 probes cover the rifampicin resistant determining region of the rpoB gene sequence
The 5 probes bind to the wild type and not the resistance mutations => 5 probes binding = no resistance detected
At least 2 of the 5 probes within 2 cycles and a positive amplification control is needed for MTB detection
1 or more of the probes must fail for rifampicin resistance to be detected
Other than MTB or Rif what does the geneXpert also determine?
It also determines the bacterial load through the Ct score
How do you interpret Ct score?
(3)
A Ct <16 (low) means there is a high amount of Mycobacterium
A Ct >28 (high) means there is a low amount of Mycobacterium
i.e. Ct is inversibly proportional to Mycobacterium load
What are the advantages of the gene xpert?
(5)
Real time PCR assay
Detects resistance to Rifampicin
Rapid
No need for Cat 3 facility
Only needs 131 CFU/ml compaired o 10^4 CFU/ml for smear
Can run pulmonary and extra-pulmonary samples
What is the overall sensitivty of the GeneXpert MTB/RIF when compared to smears
Smear + were over 98% positive on GeneXpert
Smear - were about 75% sensitive on GeneXpert
But overall specificity was nearly 100%
What is the overall sensitivty of the GeneXpert MTB/RIF for extra-pulmonary TB compared to culture
(3)
Overall sensitivty at 79 % and specificity at 97.3% when compared to culture
Sens in smear + was 99% but for smear - was 70%
Sens in children higher at 86% vs adults at 73%
What extra-pulmonary samples work better on the GeneXpert?
Sensitivity remaines over 85% for CSF, biopsies, urines, pus samples and FNAs
What extra-pulmonary samples have low sensitivity?
Pleural fluids @44%
Gastric aspirates @78%
Cavitary fluids @50.9%
What are some limitations of the Gene Xoert MTB/RIF assay?
(4)
Less sensitive for smear-neg sputum (75%)
Limited sensitivity in some extrapulmonary samples
Decreased capacity for some Rif-R mutaions
Occasional false positive Rif-R
What Rif-R mutations does the GeneXpert have a decreasede capacity to detect?
rpoB C533G mutation
-> just remember it cant detect all rpoB mutations - only 95%
What might cause false positive Rif-R calls on the Gene-Xpert?
(2)
Paucibacillary (leprosy skin samples) occasionally false positive due to delays in the real-time signal generated by probes D and E
Fase recognition as a nonfunctional rpoB F51 4F silent mutation as conferring RIF-R
What is the GeneXpert Ultra MTB/RIF assay
(3)
A new/improved assay for detection of TB and RIF-R
Uses two different multi-copy genes to detect MTBC DNA
Detects mutations within the rpoB gene for Rif-R
What genes does the GeneXpert Ultra use to detect MTBC DNA?
IS6110 and IS1081
What are the main improvements of the GeneXpert Ultra assay?
(6)
Increased cartridge capacity to hold double the amount of specimen and therefore DNA to improve sensitivity
Improvements in detecting trace amount of TB and RIF-R
Improved mutation detection chemistry - new probes
Improved sensitivity in children, HIV+ and extrapulmonary samples
Limit of detection decreased again to only 15.6 CFU/ml
Increased sensitivity for smear - specimens as well
How did the GeneXpert compare to the GeneXpert Ultra in detecting MTB meningitis in a 2020 study?
The Ultra was not superior for HIV- patients in detecting MTB in CSF with tuberculous meningitis i.e. not more sensitive in HIV- infected
Ultra was more sensitive in detecting very low or trace levels of bacteria i.e. in those on antimicrobia treatment
Both Xpert and Ultra were more sensitive in HIV infected than in HIV-
In general how does the GeneXpert compare to the Gene Ultra
(4)
The Ultra is 5% more sensitive but 3.2% less specific at detecting MTB
This may predispose to fals-positive results due to sample cross contamination
False-positives were seen when testing patients with a recent history of TB
Both sensitivity and specificity were the same for Rif-R detection
Write a note on the Gene Xpert XDR, how does it work?
A further improved GeneXpert from the ultra
It analyses melting temperatures (Tms) using sloppy molecular beacon probes (SMB) to identify mutations associated with resistance against first and second line antimicrobials
What are the main benefits of the Xoert XDR?
Doesnt just look for rifampicin resistance - looks at INH, FLQ, ETH and SLID resistance
Can differentiate between low versus high level resistance to INH and FLQ
Proved to be 94-100% sensitive and 100% specific for all drugs except for ETH when compared to sequencing
What genes does the Xpert XDR detect and what resistance do they confer?
katG -> isoniazid
inhA -> isoniazid
gyrA -> fluoroquinolones
gyrB -> fluoroquinolones
rrs -> amikacin, kanamycin
eis -> kanamycin
What genes are mutated to confer resistance against isoniazid?
katG
inhA
What genes are mutated to confer resistance against isoniazid?
katG
inhA
What genes are mutated to confer resistance against fluoroquinolones?
gyrA
gyrB
What genes are mutated to confer resistance against amikacin?
rrs
What genes are mutated to confer resistance against kanamycin?
rrs
eis
What is the new point of care device for TB?
GeneXpert Omni
What is the GeneXpert Omni?
A prototype point of care device
A single-module battery powdered platform
Equivalent sens and spec
Increased cost
What are the three steps in using a line probe assay?
DNA extraction
amplification by PCR
Reverse hybridisation of amplified nucleic acids to specific DNA probes bound on strips
Evaluation
What are the five line probe assays available?
(5)
GenoQuick MTB -> ID only
FluoroType MTB -> ID only
Genotype MTBDR/MTBDRplus - first line resistance
Genotype MTBDRsl - first and second line resistance
GenoScholar PZA-TB - first line resistance
What are some benefits and cons of line probe assays
Some can detect resistance to a broader range of 1st line and 2nd line antimicrobials
Can provide mutation specific data for common variants
The are more prone to contamination as more handling is required
What is the GenoQuick MTB line probe assay, what does it detect, how does it work?
LPA for the rapid direct detection of the MTC from pulmonary and extrapulmonary patient specimens
Detects 23S rRNA genes
Hybridisation of labelled amplicons to oligonucleotide probes arranged on a membrane strip
What does the GenoQuick MTB detect?
Detects 23S rRNA genes
How does the GenoQuick MTB work
Hybridisation of labelled amplicons to oligonucleotide probes arranged on a membrane strip
How does the GenoQuick MTB work
Hybridisation of labelled amplicons to oligonucleotide probes arranged on a membrane strip
Who makes the majority of line probe assays?
Hain Life Sciences
What does the FluoroType MTB LPA do?
What are the pros of this LPA?
Rapid detection of the MTC from pulmonary and extrapulmonary patient specimens
Its an automated version of the GenoQuick LPA
It can have a result in about 3 hours
87-100% specific
What is the GenoType MTBDRplus LPA, what does it do?
Identification of MTC
Identification of resistance to rifampicin (RMP) and/or isoniazid (INH)
Talk about the GenoType MTBDRplus LPAs ability to detect resistance
Rifampicin: Detects mutations of the rpoB gene -> can detect 96% of mutations
Isoniazid: katG gene (high level resistance) + promoter region of the inhA gene (low level resistance) -> can detect 75% of mutations
What is the KatG gene and what is it involved in?
It encodes a catalase peroxidase
This confers high levels of Isoniazid resistance
What is the inhA gene and what is it involved in?
It encodes an NADH enoyl ACP reductase
It confers low levels of isoniazid resistance
Explain how the GenoType MTBDR plus works to detect resistance
LPA has probes for both the wild type of the gene and mutated genes known to cause resistance
Susceptible strains will bind at all of the wild type probes
If resistance some of the wtprobes will be missing a signal and an additional signal will be present at the mutated probe indicating which resistance gene is present
Colorimetric assay
Talk about the GenoType MTBDRsl
These LPAs are able to detect additional resistance in fluoroquinolones, aminoglycosides, cyclic peptides and ethambutol
Prior to the Xpert XDR these were considered the best but now the XDR is definitely less complicated and preferred as its a closed system
What resistance does gyrB encode?
Fluoroquinolone resistance
What does the eis gene confer resistance against?
Aminoglycoside resistance -> kanamycin resistance
What does the emb8 gene confer resistance against?
Ethambutol resistance
What are the pros and cons of the GenoType MTBDRsl?
(5)
Sensitivity, specificity and accuracy for detection of XDR-TB was 100% -> used to rule in XDR-TB rather than rule out
Ethambutol sensitivty only 65% => not recommended for the detection of ethambutol resistance
Might have to do more than one line probe assay to encompass all resistance
Not recommended for use directly on clinical specimens
Not a closed system
What is the GenoScholar PZA-TB, how does it work?
A LPA for the rapid detection of resistance to pyrazinamide (PZA)
It taregts a 700bp fragment designed to cover the entire pncA gene
Talk about PZA resistance
High rates of PZA resistance (39%-60%) in patients wih MDR-TB and XDR-TB
What are the benefits of the GenoScholar PZA-TB?
DST for PZA is rarely performed as it requires stringent control of pH and inoculum size = LPA is a big strep forward => allows us to detect more of it
Good sensitivity of 93.2% and specificity of 91%
What PCR method is used for TB?
Targeted NGS Deeplex-MycTB kit
Talk about Targeted NGS Deeplex-MycTB kit
ultra-deep sequencing of a single 24-plex amplification of the main resistance targets
Identify the mycobacterial species + resistance
Genotypes the MBC -> not done in routine lab only in reference lab
Detects mutations across 18 genes associated with first and second line resistance
Talk about Targeted NGS Deeplex-MycTB kit
ultra-deep sequencing of a single 24-plex amplification of the main resistance targets
Identify the mycobacterial species + resistance
Genotypes the MBC -> not done in routine lab only in reference lab
Detects mutations across 18 genes associated with first and second line resistance
What are the pros and cons of the targeted NGS Deeplex-MycTB kit
Predicted 92% of resistance to first line adn 95% to second line
Issues with PYZ, ethambutol and low level rifampicin resistance
Offers species ID and resistance ID
Offers limited genotyping
