Mycobacteria: Mycobacterium tuberculosis Flashcards
True or false: TB is common in developing nations
True
What fraction of the world population is infected with TB?
1/3
How many deaths does TB cause annually?
1.4 million
What are the two main types of TB?
Respiratory TB and non-respiratory TB
Who catches TB?
Consider that it is a disease of the developing world (DDW):
(a) Close contacts of infected cases
(b) Travellers to endemic regions
(c) Individuals with weakened immune systems eg. HIV / elderly
(d) Homeless, drug abusers, alcoholics
What organisms can cause TB?
- TB is caused by Mycobacterium tuberculosis, M. bovis, M. africanum, M .microti, M. abscessus and M. canetii. M. tuberculosis accounts for 98% of infections in UK.
- Mycobacteria other than tuberculosis (atypical / MOTT) or non-tuberculous mycobacteria (NTM) can also cause infections (respiratory / non-respiratory) in people with underlying respiratory issues
Draw a table of Mycobacteria associated with human disease
What are the common resovoirs for these microbes?
M.leprae- from armadillos
M.bovis- from cows
M.avium- from birds
What are the main characteristics of the Mycobacterium species?
- Phylum
- Acid fast?
- Virulence factors
- Growth conditions
Phylum: Actinobacteria; filamentous
Acid Fast: Cell envelope contains 60% long-chain branched hydrocarbons (waxes)
Mycolic acid most abundant – virulence factor
Trehalose dimycolate (TDM) –CORD FACTOR
Slow growing: generation time 15-22 hour (cf 1 hour staphylococci)
Draw the mycobacteria cell wall
What are the main functions of trehalose dimycolate (TDM- CORD FACTOR?
(a) Reduces permeability to many molecules: Confers resistance to chemicals, stains, antibiotics
(b) Confers resistance to drying: Increased survival in environment
(c) Intracellular survival
What can be used to stain mycobacteria?
Ziehl-Neelsen (ZN) stain
State the stages of M. tuberculosis pathogenesis
Stage 1: inhalation of infectious particles: Droplet nuclei (5mm, approx 3 bacilli)
Stage 2: (7-21 days) MTB multiplies within macrophages (INTRACELLULAR); macrophages secrete IL-12 and present MTB antigen on their surface; eventually burst liberating MTB
Stage 3: IL-12 stimulates T-lymphocytes (CD4,CD8) to infiltrate; recognise MTB antigen; become activated (sensitized); CD4 release inflammatory factors -GAMMA IFN resulting in TUBERCLE formation (PRIMARY LESION)- hypoxic
Stage 4: MTB continues to multiply within inactivated / poorly activated macrophages and tubercle expands
Stage 5: Primary lesion heals: GHON FOCUS – type of granuloma (hypoxic) (dormant lesion; contains MTB; may re-activate when you become immunocompromised)
NB. IT IS THE CELL MEDIATED RESPONSE (CMI) IN ‘HEALTHY’ INDIVDUALS THAT ‘HEALS’ THE PRIMARY LESION
Draw a diagram to show the pathogeneis of M tubercuolsis
- Exposure to the source
- Aerosilation of TB DN
- Inhalation of bacteria
- Either no infection in 50% of people or bacteria reach the lungs and enter mactrophages in the other 25-50%
- Bacteria multiply on mactrophae
- Granulatomatous lesions begin to form (caseous necrosis)
- Bacteria cease to grow and lesion calcified (90%)
What are the stages of TB infection?
Stage 1: Droplet nuclei inhaled
Stage 2: (7-21 days) MTB multiplies within macrophages (INTRACELLULAR); macrophages secrete IL-12 and present MTB antigen on their surface; eventually burst liberating MTB
Stage 3: IL-12 stimulates T-lymphocytes to infiltrate; recognise MTB antigen; become activated (sensitized) and start to release inflammatory factors (eg. gamma IFN); TUBERCLE formation (PRIMARY LESION)
Stage 4: MTB continues to multiply within unactivated / poorly activated macrophages and tubercle expands
Stage 5: Primary lesion heals: GHON FOCUS (dormant lesion; contains MTB; may re-activate)
NB. IT IS THE CELL MEDIATED RESPONSE (CMI) IN ‘HEALTHY’ INDIVDUALS THAT ‘HEALS’ THE PRIMARY LESION
What is this?
Ghon focus
Produce a schematic to show the stages of TB infection
What are the characterstics of a Ghon focus?
Infected , inflamed, lymph node
What are the possible outcomes of a Ghon focus in a immunocompetent (healthy) host?
Cell Mediated Immunity (CMI) prevents spread of M. tuberculosis, minimal / no symptoms in 90% of hosts
- MTB remain LATENT (latent TB)
- REACTIVATION MAY OCCUR
Give examples of immunocompromised hosts
Young / old / HIV / transplant
What are the possible outcomes of a Ghon focus in a immunocompromised (healthy) host?
- Primary focus worsens; pneumonia develops (Lower respiratory tract infection)
- Systemic dissemination (lymph nodes, meninges, upper parts of the lung)
- Symptoms result from host CMI response: chronic inflammation (M. tuberculosis has no endotoxin / exotoxin
Describe Secondary TB (reactivation of latent TB)
- Endogenous - reactivation of initial infection; commonly within 2 years but may occur any time after
- Associated with any impairment of CMI (steroid therapy, immunosuppresive drugs, cancer, HIV) and local disturbance of dormant tubercules
- Caseous (cheesy) necrotic lesions develop which liquefy and discharge contents into bronchi:
(a) well –aerated environment
(b) distribution to other parts of lung
(c) contents of caseous lesions are coughed up becoming infective droplet nuclei
* TB pneumonia may result
What is this?
Caseous lesions associated with 2O TB
What is this?
Widespread distribution associated with 2O TB
What is the link between Mycobacteria and AIDS?
CMI response important in TB infection. Depletion of CD4+ helper cells (in HIV) impairs CMI.
2/3 AIDS patients in sub-Saharan Africa have TB; similar picture occurring in developed nations:
- Patients prone to rapid primary infection and reactivation of previous infection
- Advanced AIDS patients (<50 CD4 cells/µl) are very susceptible to M. avium-intracellulare: environmental
- Disseminated M. avium Complex (MAC) infection: chronic, fever, wasting, multiple organ involvement, death
Why is TB and HIV sometiemes referred to as the twin epdemics?
- 1/3 of AIDS patients worlwide have TB
- 2/3 of AIDS patients in subsaharan africa have TB and HIV
What percentage of people with TB are asymptomatic?
90%
What percentage develop primary tuberculosis and experience symptoms in which reactivation of latent (dormant) TB may occur?
10%
Name some lower respiratory tract infection associated with TB:
- cough (sputum with blood)
- significant weight loss
- night sweats
- Fatigue
- fever
May spread to other body parts (Miliary TB):
- Meningitis
- Septicaemia
- kidney infection
- joint infection; Pott disease
Describe the clinical diagnosos of TB
- Patient presenting with symptoms
- Radiological changes on chest X –Ray (ghon fous identified)
- Tuberculin Skin Test (TST); ‘Mantoux’ test (traditional) – Injection with Purified Protein Derivative (PPD) which elicis immune response, you get a larger injuration if you have been exposed with the tb vaccine but not work if you have had the BCG vaccine.
Laboratory diagnosis- non culture techniques to diagnose TB
- Interferon Gamma Release Assay (IGRA)
- Molecular detection in clinical samples (Nucleic Acid Amplification; PCR)
- Microscopy (low resolution for TB need a high bacterial load)
Laboratory diagnosis- culture techniques to diagnose TB
What is the gold standard?
(gold standard method) - MGIT
Which clinical samples are used to establish a laboratory diagnosis for TB?
- Early morning sputum (x3)
- Renal TB: Complete early morning urine (EMU X3)
- CSF (meningitis)
- Lymph node biopsy
- Blood / Bone marrow aspirate
- Whole blood (for IGRA test)
True or False: Mycobacterium tuberculosis is a category 2 pathogen
False
Mycobacterium tuberculosis is a category 3 pathogen. It is very infectious, you need to take serious precautions when handling it. Class 1 safety cabinet.
Non-culture techniques:
Describe Interferon Gamma Release Assay (IGRA)
THEORY: A person’s T-cells that previously were sensitized to TB antigen (i.e TB infection) produce high levels of IFN-gamma when re-exposed to the same mycobacterial antigen.
Huge amount more IFN-gamma produce if previously exosured compare to first exposure
Non-culture techniques:
Explain Interferon Gamma Release Assay (IGRA)
- TB diagnosis: Blood sample from a patient with possible active or latent TB is re-exposed to specific mycobacterial antigens
- Antigens utilised are encoded in region of difference (RD)1 –a genomic sequence absent from most MOTT (eg BCG vaccine, environmental mycobacteria)
RD1 encoded antigens:
(a) Early Secretary Antigenic Target (ESAT-6)
(b) Culture Filtrate Protein (CFP-10)
Non-culture techniques:
Interferon Gamma Release Assay (IGRA)
Which commercial kits are available:
(a) QuantiFERON-TB Gold; INF-gamma secretion measured
(b) T-SPOT.TB; INF-gamma secreting T-cells enumerated
Non-culture techniques: Microscopy
Describe the Ziehl-Neelson stain (ZN) test
- Lower limit of detection of ZN stain is 5 x 103 orgs/mL
- Light microscopy x100 objective (X 1000 magnification
- Not very sensitive (need a high bacterial load)
Non-culture techniques: Microscopy
Describe the Fluorescent auramine stain
- Increases sensitivity of microscopy for TB diagnosis
- Fluorescent microscopy x 40 objective (X 400 magnification; increased sensitivity)
- Positive results within 1 hour: rapid diagnosis if positive
- Negative smears do not rule out TB (low numbers of microorganisms in samples)
- Overall sensitivity of microscopy: <50%
NEWS JUST IN:
What is TB-REaD?
- Uses enzyme endogenous in TB (BLAc) a beta lactamase
- Custom chemical probe that interacts with this enzyme specific to TB bacteria
- Rapid Fluorescent signalling system once beta lactamase hydrolyses the probe
Compare methods of TB diagnosis
Respiratory TB: Culture (gold standard) Describe Sample Preparation eg. SPUTUM (non sterile)
(a) Liquefaction of sputum (‘sputasol’, N-acetyl cystine)
(b) Concentration (centrifugation)
(c) Decontamination: 4% NaOH (kills off everything but TB)
NB. ‘Sterile’ samples eg. Blood do not need decontamination
Respiratory TB: Culture (gold standard) Describe Sample Preparation eg. SPUTUM (non sterile)
Describe solid culture
eg. Lowenstein-Jensen (LJ) slopes
- whole eggs; salts; glycerol; potato flour
- malachite green; penicillin; nalidixic acid
- Incubation: 37oC for up to 8 weeks (M. tuberculosis)
Respiratory TB: Culture (gold standard) Describe Sample Preparation eg. SPUTUM (non sterile)
Describe broth culture
eg. Bactec mycobacterial broth
- Semi-Automated systems: BACTEC
Describe the full Identification of M. tuberculosis Colonies
- Colonial appearance
- ZN positive colonies
- Biochemical characteristics (see table):
can take up to 4 weeks to get result
How can M.tuberculosis be differentiated from other mycobacteria
M.tuberculosis is Niacin and nitrate reductase positive.
Full Identification of M. tuberculosis Colonies
Rapid ID molecular probes
ID in 1-2 days
(a) species specific DNA probes
(b) ribosomal rRNA based probes (10-100x more sensitive than DNA probes)
Where does the UK Recommendations Treatment of Respiratory TB come from?
- British Thoracic Society (BTS)
- National Institute for Health and Care Excellence (NICE)
What is the recommended management of active TB?
Six-month course of treatment in 2 phases:
ISONIAZID, RIFAMPICIN, PYRAZINAMIDE, ETHAMBUTOL
(a) Treatment supervised by trained physician
Describe methods employed to improve patient compliance
Directly observed therapy (DOT) following patient risk assessment. DOT should be considered for patients having adverse factors on risk assessment eg. street/shelter dwelling homeless, alcoholics
These guidelines are aimed at:
(a) Successfully eliminating the infection
(b) Preventing drug resistance amongst strains of TB
In the UK what percentage of strains are resistant to one or more of the antibiotics
6-8%
MDR-TB
Resistant to at least isoniazid and rifampicin; the two most powerful anti-TB drugs
Drug resistance and MDR in TB arises due to:
How can we avoid MDR in TB?
(a) Administration of improper treatment regimens by health care workers
(Ans: Trained TB physicians- lots of susceptibility testing)
(b) Failure to ensure that patients complete the whole course of treatment
(Ans: Directly observed therapy DOT)
Treatment of active TB- Initial Phase (2 months):
Isoniazid, Rifampicin, Pyrazinamide, Ethambutol
Treatment of active TB- Continuation Phase (4 months):
Isoniazid, Rifampicin
Describe the treatment of active TB
Fixed dose combination tablets are available and are recommended as first choice
Describe combination drug preparations
- Aid in patient compliance and therefore the successful treatment of TB - reduce pill burden
- ‘Rifater’
initial phase:
isoniazid / rifampicin / pyrazinamide
- ‘Rifinah 300’ or ‘Rimactazid 300’
continuous phase:
isoniazid / rifampicin
Not used predominanatly in the UK
Treatment of TB: Isoniazid
What is the MOA and side effects?
How does resistance to isoniazid occur?
Inhibits cell wall formation
- MOA:
Isoniazid is a prodrug which is converted to isonicotinic acid-NADH complex. Binds to InhA which facilitates mycolic acid synthesis
- Side effects
Hepatitis
Peripheral
neuropathy
Resistance
Short: Isoniazid is activated by KatG (catalase peroxidase) gene which binds and inhibits an enzyme essential to the production of mycolic acid. Stops mycol
Long: INH (isniazid) resistance commonly occurs due to mutations in the katG gene or the inhA regulatory regions. katG encodes catalase peroxidase, an enzyme that converts INH to its biologically active form. As mutations in katG, particularly at codon 315, confer high-level INH resistance, INH is ineffective for the treatment of Mycobacterium tuberculosis
Treatment of TB: Rifampicin
What is the MOA and side effects?
Inhibits nucleic acid synthesis
- MOA
Binds to RNA polymerase thus
blocking mRNA synthesis and subsequent nucleic acid synthesis
- Side effects
Hepatitis
Stains body fluids orange (sweat, tears, urine etc)
Treatment of TB: Pyrazinamide
What is the MOA and side effects?
Cell acidification
- MOA
Converted to pyrazinoic acid by pyrazinamidase (decreased pH). Inhibits ribosome
- Side effects
Hepatitis
Treatment of TB: Ethambutol
What is the MOA and side effects?
Inhibits cell wall formation
- MOA:
Binds to arabinosyl transferases disrupting formation of cell wall components (AG)
- Side effects
Ocular toxicity -reversible
Multi-drug resistant TB (MDR-TB)
- SHREZ (Streptomycin + isonicotinyl Hydrazine + Rifampicin + Ethambutol + Pyrazinamide) with moxifloxacin + cycloserine
- Sensitivity testing
- Case by case treatment strategy: susceptibility vs. toxicity
Extensively drug resistant TB (XDR-TB)
MDR-TB + a quinolone + (at least one of the second-line TB injectables (kanamycin, capreomycin, amikacin)
Totally drug resistant TB (TDR-TB)
Where has it been identified?
- Identified in India, Iran and Italy (2009-2012)
- Not recognised by WHO
Key Points: Tuberculosis
- One-third of the world’s population is thought to be infected with TB and the incidence of TB is rising (linked with HIV increase).
- Immunocompromised patients are significantly more at risk from developing disease following exposure.
- Primary and secondary (reactivation) pulmonary TB exist.
- Accurate diagnosis facilitates appropriate treatment which is essential to control the spread of TB, minimise resistance and reduce incidence.
- Treatment is bi-phasic, aggressive and lasts for 6 months; consider DOT where necessary.
- Side effects are associated with treatment of TB: Rifampicin, isoniazid and pyrazinamide are hepatotoxic. Ethambutol may have ocular toxicity.
Does MTB have edo/exotoins?
No
Describe MDIT
Mycobacerium indicator test
What is MGIT?
Mycobacteria Growth Indicator test
- A fluorescent compound is embedded in silicone on the bottom of 16 × 100 mm round bottom tubes.
- The fluorescent compound is sensitive to the presence of oxygen dissolved in the broth.
- Initially, the large amount of dissolved oxygen quenches emissions from the compound and little fluorescence can be detected.
- Later, actively respiring microorganisms consume the oxygen and allow the fluorescence to be detected.
Tubes are filled with samples in the broth and continuously incubated at 37°C.
- The tubes are monitored for increasing fluorescence to determine if the tube is instrument positive; i.e., the test sample contains viable organisms.
How can TB treatment time poteintially be reduced?
What would the main benefits be?
- Tuberculosis treatment for people without drug resistance can be shortened from six months to four months by replacing two drugs in the standard regimen with newer agents (high-dose rifapentine and moxifloxacin), results from a large international trial show.
- A shorter course of treatment would improve adherence and reduce costs for the health system and people with TB. In particular, it would enable directly observed treatment to be completed quicker and would allow people with TB to return to work quicker, reducing the household impact of TB.
