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2.2 Microbiology > Denise - Mycobacterium > Flashcards

Denise - Mycobacterium Flashcards

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1
Q

Write about the TB WHO 2022 Report

A

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

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2
Q

Write about Mycobacterium species

A

Acid fast cell wall
>90 species

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3
Q

What is meant by acid fast cell wall

A

Organism is stained with a basic dye but cannot be decolourised subsequently with dilute acids

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4
Q

What does MTC mean

A

Mycobacterium tuberculosis complex

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5
Q

What does NTM mean

A

Non tuberculosis mycobacteria

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6
Q

What does MOTT mean

A

Mycobacteria other than tuberculosis

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7
Q

Write about the cell wall of TB structure

A

Peptidoglycan and complex lipids
High lipid content (up to 60%)
Hydrophobic and waxy

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8
Q

What is the function of the TB cell wall

A

Major determinant of virulence
Resistance
Decolourisation
Disinfectants
Antimicrobials
Immune Cells

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9
Q

What are the components of the TB cell wall lipid fraction

A

Mycolic acids
Cord factor
Wax-D

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10
Q

Write about tuberculosis

A

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

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11
Q

What are the symptoms of TB

A

Chronic cough
Haemopytsis
Drenching night sweats
Weight loss
Pyrexia

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12
Q

Write about TB epidemiology

A

10.4 million cases a year
1.8 million deaths
Most common cause of death in HIV
>4million never diagnosed and treated

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13
Q

Write about TB resistance

A

600,000 rifampicin resistant cases

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14
Q

How many cases in ireland in 2019

A

267

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15
Q

What are the risk factors for TB

A

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

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16
Q

What are the four stages of pathogenesis in TB

A

Primary tuberculosis
Progressive primary active infection
Latent-dormant tuberculosis
Seconday reactivation TB

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17
Q

What is primary tuberculosis

A

TB skin test negative
Radiograph negative

10% progress to progressive primary (active) infection

90% progress to latent-dormant tuberculosis

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18
Q

What is progressive primary active infection

A

TB skin test positive
Radiograph positive
Sputum positive

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19
Q

What is latent dormant tuberculosis

A

TB skin test positive
Radiograph negative for active disease

90% progress to no disease
10% progress to secondary reactivation TB

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20
Q

What is secondary TB

A

TB skin test positive
Radiograph positive
Sputum positive

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21
Q

What are the clinical findings of TB

A

Tuberculin skin test positive
IFN assay positive
Chest X ray positive

22
Q

What three microbiological investigations are carried out for TB

A

Microscopy
NAAT
Culutre

23
Q

What specimens are seen in TB

A

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

24
Q

Write about TB specimen processing

A

Hazard group 3 pathogen
Biosafety/Containment level 3 laboratories
Environmental persistence
Precautions to avoid aerosol formation and inhalation

25
Q

What three safety procedures are in place to prevent TB

A

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

26
Q

What three things are done at specimen processing

A

Grade the appearance

Mucolyse (Homogenise)

Decontaminate

27
Q

How are specimens graded

A

Bloodstained
Purulent
Mucosalivary

28
Q

How are samples decontaminated

A

Required to remove commensals
Kill most commensals and preserve most mycobacteria
2% NaOH
Contamination rate 2-5% required

29
Q

Write about the direct smear microscopy of TB

A

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

30
Q

Write about the acid fast stain

A

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

31
Q

What is the limit of detection for TB

A

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

32
Q

Write about TB culture

A

Solid media
Lowenstein-Jensen medium slopes
Grows well on this medium

33
Q

What does the Lowenstein-Jensen slopes contain for M tuberculosis

A

Egg
Malachite
Green
Glycerol

34
Q

Write about the Lowenstein-Jensen medium for M. bovis

A

Eggs
Malachite
Green
Pyruvate

35
Q

What cocktail is used for TB

A

PANTA cocktail

36
Q

What is containes in the PANTA cocktail

A

Polymyxin
Amphotericin
Nalidixic acid
Trimethoprim
Azlocillin

37
Q

Write about culture liquid medium for TB

A

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

38
Q

What automated detection systems are used for TB

A

Mycobacterial growth indicator tube (MGIT)
Bactec 90000
MB BacT

39
Q

Write about automated detcetion

A

An automated fluorescnet detection method
Automated reading technology

40
Q

What is the protocol behind automated detection of Blood cultures

A

 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

41
Q

What is microscopy used for

A

Cording morphology of M. TB
Non-cording of non M. TB
AuromineO stain
ZN stain

42
Q

What molecular methods are used

A

MGIT TBcAssay

43
Q

Write about molecular detection

A

Enclosed RT-PCR
GeneXpertMTB/RIF Ultra Assay
Rpo gene

44
Q

Write about the GeneXpert MTB/RIF assay

A

 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

45
Q

Write about line probe assays

A
  1. DNA extraction
  2. amplification by PCR
  3. hybridisation to specific DNA probes bound on strips
  4. evaluation

GenoType MTBC kit Differentiation of the Mycobacterium TB complex

GenoType Mycobacterium CM

Suitable for direct detection clinical specimens and culture

46
Q

Write about susceptibility testing

A

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

47
Q

Write about susceptibility system

A

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.

48
Q

Write about targeted NGS Deeplex- MycTB GenoScreen

A

 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

49
Q

What does MDR mean in TB

A

Resistant to rifampicin and isoniazid
250k cases a year

50
Q

What does XDR mean in TB

A

Resistant to rifampicin and isoniazid plus 1 fluoroquinolone, 1 aminoglycoside

25k cases worldwide

51
Q

What is the TB vaccine called

A

BCG vaccine

52
Q

Where is the TB lab and what is done here

A

Irish Mycobacteria Reference Laboratory IMRL at St James Hospital

Identification
Antimicrobial susceptibility testing
Epidemiological strain typing

2.2 Microbiology (60 decks)

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