Denise - Mycobacterium Flashcards
Write about the TB WHO 2022 Report
Leading cause of death from a single infectious agent
10 million cases
1.4 million deaths
8.2% of cases were people with HIV
500,000 people developed rifampicin-resistance
78% had multidrug-resistant TB
COVID-19 threatens to reverse recent progress in reducing the global burden of TB disease
Write about Mycobacterium species
Acid fast cell wall
>90 species
What is meant by acid fast cell wall
Organism is stained with a basic dye but cannot be decolourised subsequently with dilute acids
What does MTC mean
Mycobacterium tuberculosis complex
What does NTM mean
Non tuberculosis mycobacteria
What does MOTT mean
Mycobacteria other than tuberculosis
Write about the cell wall of TB structure
Peptidoglycan and complex lipids
High lipid content (up to 60%)
Hydrophobic and waxy
What is the function of the TB cell wall
Major determinant of virulence
Resistance
Decolourisation
Disinfectants
Antimicrobials
Immune Cells
What are the components of the TB cell wall lipid fraction
Mycolic acids
Cord factor
Wax-D
Write about tuberculosis
Highly contagious
Aerosolised droplets
Mainly affect lungs: pulmonary TB
Can affect any organ: extra pulmonary TB
Can spread via the bloodstream and lymphatics - may lead to miliary TB
Progressive disseminated haematogenous TB
1/3 people latently infected with latent TB - asymptomatic carriage
10% may develop active infection - constant infectious reservoir
What are the symptoms of TB
Chronic cough
Haemopytsis
Drenching night sweats
Weight loss
Pyrexia
Write about TB epidemiology
10.4 million cases a year
1.8 million deaths
Most common cause of death in HIV
>4million never diagnosed and treated
Write about TB resistance
600,000 rifampicin resistant cases
How many cases in ireland in 2019
267
What are the risk factors for TB
Exposure to person with active TB
Large infecting dose
Age and gender
Health- leukaemia, hodgkins disease, diabetes melitus, alcoholism, IV drug abuse etc
Malnutrition, low body weight, vitamin D deficiency
smoking
Occupation
Immunosuppression
Stress
What are the four stages of pathogenesis in TB
Primary tuberculosis
Progressive primary active infection
Latent-dormant tuberculosis
Seconday reactivation TB
What is primary tuberculosis
TB skin test negative
Radiograph negative
10% progress to progressive primary (active) infection
90% progress to latent-dormant tuberculosis
What is progressive primary active infection
TB skin test positive
Radiograph positive
Sputum positive
What is latent dormant tuberculosis
TB skin test positive
Radiograph negative for active disease
90% progress to no disease
10% progress to secondary reactivation TB
What is secondary TB
TB skin test positive
Radiograph positive
Sputum positive
What are the clinical findings of TB
Tuberculin skin test positive
IFN assay positive
Chest X ray positive
What three microbiological investigations are carried out for TB
Microscopy
NAAT
Culutre
What specimens are seen in TB
Non sterile sites:
- Pulmonary (sputa, BAL, bronchial washings)
- renal (urine)
Sterile sites
- pleural fluids, joint fluids, CSF
- tissue
Poor specimen
- poor smear and culture results
- no organism = no identifcation, suceptiblity or typing results
Write about TB specimen processing
Hazard group 3 pathogen
Biosafety/Containment level 3 laboratories
Environmental persistence
Precautions to avoid aerosol formation and inhalation
What three safety procedures are in place to prevent TB
Centrifuges pose the risk of aerosols and breakages and therefrore sealed buckets should be used
Discared material may be preliminarily disinfected in hypochlorite soluteion and then autoclaves
Lab perosnal should be Mantoux tested and given the BCG vaccine if necessary
What three things are done at specimen processing
Grade the appearance
Mucolyse (Homogenise)
Decontaminate
How are specimens graded
Bloodstained
Purulent
Mucosalivary
How are samples decontaminated
Required to remove commensals
Kill most commensals and preserve most mycobacteria
2% NaOH
Contamination rate 2-5% required
Write about the direct smear microscopy of TB
Acid fast stain
Retain the primary stain when the smear is decolourised with an acid alcohol solution
Auramine-O stain
Dirext ZN
Rapid, cost effect
Shoulf be available within one working day
Poor sensitivity
Write about the acid fast stain
Sputum smear AFB positive
- indicates TB
Smear negative
- inficates patient less infectious
Used to determine whether isolation is required
Influences the extend of contact tracing
Used to monitor treatment
What is the limit of detection for TB
Smear microscopy = 1000 CFU/ml
Culture (gold standard) = 10-100 CFU/ml
GeneXpert MTB/RIF assay: = 131 CFU/ml
GeneXpert MTB/RIF Ultra assay = 15.6 CFU/ml
Write about TB culture
Solid media
Lowenstein-Jensen medium slopes
Grows well on this medium
What does the Lowenstein-Jensen slopes contain for M tuberculosis
Egg
Malachite
Green
Glycerol
Write about the Lowenstein-Jensen medium for M. bovis
Eggs
Malachite
Green
Pyruvate
What cocktail is used for TB
PANTA cocktail
What is containes in the PANTA cocktail
Polymyxin
Amphotericin
Nalidixic acid
Trimethoprim
Azlocillin
Write about culture liquid medium for TB
Middlebrook 7H9 broth
A liquid growth medium specially used for culture of M, TB
Supplemented with glycerol for M. TB
Liquid culture is more sensitive and shorter time needed for positive
Contamination and mixed cultures are difficult
Used for automated detection
What automated detection systems are used for TB
Mycobacterial growth indicator tube (MGIT)
Bactec 90000
MB BacT
Write about automated detcetion
An automated fluorescnet detection method
Automated reading technology
What is the protocol behind automated detection of Blood cultures
A fluorescent oxygen sensor is embedded in the base of the tube
Detects any decrease in O2 dissolved in broth
Oxygen sensor will emit light when exposed to UV
Actively respiring organisms consume O2
Reduction in O2 is detected by machine
Machine flags tube as positive
What is microscopy used for
Cording morphology of M. TB
Non-cording of non M. TB
AuromineO stain
ZN stain
What molecular methods are used
MGIT TBcAssay
Write about molecular detection
Enclosed RT-PCR
GeneXpertMTB/RIF Ultra Assay
Rpo gene
Write about the GeneXpert MTB/RIF assay
Automated NAAT
Closed system
Minimal hands-on technical time
Rapid results (<2 hours)
WHO endorsed
Dec 2010
Detects MTB and RIF resistance
* Limit of detection:
Xpert MTB/RIF Assay: 131 CFU/ml
Smear Microscopy: <=104 CFU/ml
Culture (Gold std): 10 -100 CFU/ml
GeneXpert MTB Ultra
Write about line probe assays
- DNA extraction
- amplification by PCR
- hybridisation to specific DNA probes bound on strips
- evaluation
GenoType MTBC kit Differentiation of the Mycobacterium TB complex
GenoType Mycobacterium CM
Suitable for direct detection clinical specimens and culture
Write about susceptibility testing
Conduction when patient is first found ot have positive culture for TB
Determines which drugs kill tubercle bacilli
Molecular methods hours-1 day
- geneXpert
- GenoTypeMycobacterium MTBDR plus
- identification of the M TB complex
- resistance to rifampicin and/or isoniazid
Liquid Media BACTEC MGIT 7-10 days
Solid media LJ MB 3-4 weeks
Write about susceptibility system
MGIT 960 system
Liquid culture based semi automated detection method
7-10 days detection time
A known concentration of antimicrobial is added to a MGIT tube, along with the specimen, and growth is compared with a antimicrobial -free control
If the antimicrobial is active growth will be inhibited and fluorescence will be suppressed in the antimicrobial -containing tube
the antimicrobial -free control will grow and show increasing fluorescence.
If the isolate is resistant to the antimicrobial, growth and an increase in fluorescence will be evident in both the antimicrobial -containing and the antimicrobial - free tube.
Write about targeted NGS Deeplex- MycTB GenoScreen
Ultra-deep sequencing of a single, 24-plex amplification of the main resistance targets
Identify the mycobacterial species
Genotyping targets-type strains of the M.tuberculosis complex
Detect mutations in 18 gene regions associated with resistance to first- and second-line drugs
What does MDR mean in TB
Resistant to rifampicin and isoniazid
250k cases a year
What does XDR mean in TB
Resistant to rifampicin and isoniazid plus 1 fluoroquinolone, 1 aminoglycoside
25k cases worldwide
What is the TB vaccine called
BCG vaccine
Where is the TB lab and what is done here
Irish Mycobacteria Reference Laboratory IMRL at St James Hospital
Identification
Antimicrobial susceptibility testing
Epidemiological strain typing
