Mycobacteria: Mycobacterium tuberculosis Flashcards
What are Mycobacteria?
Describe its properties?
- Unicellular rod-shaped micro-organisms
- obligate aerobes
- non-motile
- non-capsulated
- non-sporulating
- 2-4 μm length X 0.2-0.5 μm width
- Many species of Mycobacterium exist; only a few cause disease in humans
- Many important species are SLOW GROWING
(generation time: 15-20 hrs)
Cell wall comprises HIGH LIPID CONTENT (unlike Gram positive / negative bacteria)
Describe a Gram positive cell wall
Cytoplasmic membrane followed by a thick layer of peptidoglycan leafleted with teichoic acid and lipoteichoic acid
Describe a Gram negative cell wall
Outer membrane (with LPSand lipid A) and cytoplasmic membrane sandwiching a thin layer of peptidoglycan
Mycobacteria: Describe its unique Cell Wall Structure
- Features of cell wall:
- (60% lipid)
- Inner membrane followed by a large periplasmic space leafleted with LAM and towards the end the LAM is anchored to a pepdioglycan layer
- cental component of the cell wall is thearabinogalactan-peptidoglycan layer
- High glycolipid content eg. MYCOLIC ACID (unique to mycobacteria) in the outer mebrane
- Other lipids include: surface acyl lipids; lipoarabinomannan (LAM); phosphatidy inositol mannosides (PIM); cord factor-Trehalose Dimycolate (TDM)(sugar with 2 mycolic acids)
- TDM aids intracellular survival of mycobacteria→stops lysosome from fusing with phagosome
Why is the cell wall of myobacteria important medically?
- It promotes Intracellular survival (prevents phagosome fusing with lysosome)
- Confers resistance (eg. many antimicrobials, heat, chemicals, drying, STAINS)
- Mycobacteria are ACID-FAST
- Special stains required to visualise Mycobacteria
What stain is used for acid-fast bacteria?
Ziehl-Neelson Stain
Describe the Ziehl-Neelson Stain for Acid-Fast bacteria
- Stain with hot concentrated carbol fuchsin and (all bacterial cells stained pink at this stage)
- De-stain with 1% acid-alcohol (hydrochloric acid / ethanol) OR 20% H2SO4 (mycobacterial cells remain pink, all others colourless)
- Counterstain with methylene blue (mycobacterial cells remain pink, all others bacteria and cells eg WBC stain blue)
Infections Caused by Mycobacteria: Tuberculosis (TB)
What species of myobacterium is this disease caused by?
M. tuberculosis
What are the symptoms of TB?
- Often asymptomatic
- pneumonia-like presentation (weight loss, temperature, cough, blood in sputum)
Infections Caused by Mycobacteria: Tuberculosis (TB); acquired from consumption of unpasteurised milk
What species of myobacterium is this disease caused by?
This is caused by M. bovis
Infections Caused by Mycobacteria:
Disseminated TB in immunocompromised patients eg. HIV
What species of myobacterium is this disease caused by?
M. avium intracellulare
Non-tuberculosis mircrobacterium (NTM)
What the symptoms of Disseminated TB in immunocompromised patients eg. HIV?
Lung infection and spread to bones, joints, blood, renal system, meninges; frequently results in death
Which disease is cauesd by M. leprae (Hansen’s bacillus)?
Leprosy
What are the symptoms of leprosy?
Folded lesions on face and limbs
Disfiguration
Loss of peripheral nerves
Secondary infection
Tuberculosis: Describe the beginnings
- 1720: Benjamin Marten described ‘Consumption’; wonderfully minute living creatures; A New Theory of Consumption published
- 1882: discovered the cause of ‘consumption’; caused by an infectious agent; Koch’s bacillus (M. tuberculosis)
- Granville’s Mummy (600BC): First ancient Egyptian mummy to be subjected to a scientific autopsy (1825); incorrect diagnosis-ovarian tumour, found mycolic acid to disprove this.
- 21st century (UCL); TB DNA identified (lung, femur, gall bladder) in Granville’s Mummy – ‘a wrong diagnosis’ by Granville
State 21st Century Tuberculosis figures
Worldwide Figures
- 1/3 of world population predicted to have TB
- 8.4 million new cases per year
- 1.5 million deaths per year
The Fall and Rise of TB on the Rise in UK:
State UK historical trends and annual figures
1950: 50,000
mid-1980s: 6,000
1987-1990: 7,000
2010: 9,000
now: 6000
State some predisposing factors of TB infection
- HIV infection is the MAIN predisposing factor for TB infection. 10 percent of all HIV-positive individuals have TB (400-times the rate associated with the general public)
- Close contact with large populations of people, i.e., schools, nursing homes, dormitories, prisons, etc
- Poor nutrition
- iv drug use
- Alcoholism
TB Infection: Descibe the properties of droplet Nuclei
What releases droplet nuclei?
- TB spreads via Droplet nuclei (5mm, approx 3 bacilli- minimum infective dose)
- Aerosolised:
Talking for 5 min: 3,000
Coughing: 3,000
Sneezing: 3,000 up to 10 feet away
(a) remain suspended for hours; environment is infectious after infected individual has left
(b) small size allows for bypass of mucociliary lining
* Coughing, talking, sneezing releases DN
Explain the stages of TB Infection
Stage 1: Droplet nuclei inhaled
Stage 2: (7-21 days) Microbacterium tuberculosis-MTB multiplies within (alveolar) 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 or granuloma) provides protection of the host from MTB and MTB from the host. MTB can secrete immunomodulatory and antiinflammatory factors
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) formed in the lung, isolation site if MTB
NB. IT IS THE CELL MEDIATED RESPONSE (CMI) IN ‘HEALTHY’ INDIVDUALS THAT ‘HEALS’ THE PRIMARY LESION
Describe the TB Granuloma
Once the immune system has successfully encapsulated the infection
- Necrotic infected macrophages in the center that has burst and release the bacteria
- Te bacteria use the nutrients from the necrotic macrophages for their own metabolism but are under hypoxic conditions
- Surrounding this are macrophages that have become epithelioid (outside of bacteria, interior of granuloma)
- T and B cell that form the wider protective barrier
Draw a diagram to show the stages of TB Infection
- Exposure to source
- Aerosolisation of TB
- Inhalation of bacteria
- Bacteria reach lungs + enter macrophages (25-50%)
- Bacteria multiply in macrophages
- Granulomatous lesions begin to form (caseous necrosis)
- Latent or active infection
Compare latent to active TB infection
After granulomatous lesions begin to form, bacteria can cease to grow and lesion calcifies (90%)→ LATNET TB INFECTION
Alternatively, the lesion liquefies and bacteria can spread to blood/organs→ACTIVE TB INFECTION Bacteria may be coughed up in sputum. This can lead to death
TRUE or ALSE: 90% of individuals with TB are asymptomatic
TRUE
Healthy cell mediated immune response (CMI) keeps infection under control
10% of individuals with TB develop primary tuberculosis and experience what symptoms?
Lower respiratory tract infection:
- cough (sputum with blood)
- weight loss
- night sweats
- fatigue
- fever
May spread to other body parts:
- meningitis
- blood poisoning
- kidney infection
- joint infection
Patients are hyper-sensitised ie. cell mediated immunity (T-lymphocytes) ready to attack re-exposure to M. tuberculosis
What are the symptoms of secondary tuberculosis?
- (a) associated with any impairment of cell mediated immunity-CMI (steroid therapy, immunosuppressive drugs, cancer chemotherapy, old age, HIV)
- (b) dormant PRIMARY focus becomes necrotic (cheesy) and liquefies distributing 1000s M. tuberculosis into the lungs
- (c) widespread distribution of M. tuberculosis in the lungs (and possibly throughout the body)
What is the appropriate clinical sample in laboratory Diagnosis of Pulmonary (respiratory) TB?
Sputum (or bowel) sample
Sputum is processed in a class 3 laboratory under a class 1 safety cabinet
TRUE or FALSE: M. tuberculosis is a category 2 microorganism
FALSE
M. tuberculosis is a category 3 microorganism
What is a category 3 microorganism?
A biological agent that can cause severe human disease; serious hazard to employees; risk of spread in community; effective treatment / prophylaxis
Describe the microscopy of Sputum
- Microscopy of smears of sputum stained by ZN (light microscopy)
- View using x100 objective (=1000x magnification)
- Examine for acid-fast (ZN positive bacteria)
- Need approx 10,000/mL (high bacterial load) of sputum to visualise one organism
Describe how to culture sputum
- Culture sputum on specialised agar: Lowenstein-Jensen (LJ slopes) –different from other selective microbiological agars
- Contains: egg, glycerol, minerals, potato flour, antibiotics and malachite green
- Incubate at 37oC for up to 12 weeks
- Examine for LJ slopes for typical colonies of M. tuberculosis (wrinkly/crusty orange colonies)
Describe Mycobacterium tuberculosis colonies
- Generally grows in 4-6 weeks
- Yellow / buff coloured colonies;irregular in shape
- Colonies are ZN positive
- Form distinctive serpentine cords when stained (caused by TDM). This observation was first made by Robert Koch (Cord factor: trehalose 6, 6’ dimycolate - a glycolipid found in the cell walls of mycobacteria - causes the cells to grow in ‘serpentine’ cords
- Cord factor: Virulence factor; toxic to mammalian cells and inhibits white blood cell migration
Explain TB treatment
How does it differ for drug sensitive and drug resistant TB?
TB is difficult to eradicate; six month treatment regime (for drug sesnsitve TB); toxic anti-mycobacterial drugs
Intravenous antibiotics for drug resistant TB (for up to 18 months)
Combination therapy:
- Isoniazid
- Rifampicin (stains sweat and urine red)
- Pyrazinamide
- Ethambutol
All taken for the first 2 months (initiation phase)
For the remaining 4 months (continuation phase) only
Isoniazid and Rifampicin are taken
What are the main side effects of drugs used to treat TB?
Side effects include: hepatitis (inflammation of the liver); gastrointestinal disturbances
Describe the methods of TB prevention
BCG Vaccination (1953) (Bacille Calmette- Guerin):
- M. bovis BCG is a live ‘attenuated’ (reduced) form of M. bovis obtained by serial passage (230 passages over glycerinated bile potato medium) in vitro untill it lost the ability to infect a host
- 50-80% effective; lasts 10-15 years (RD1 region was mutated sufficiently so no longer active in the genome)
- 1953-2005: BCG universal vaccination programme delivered through schools
2005-present: Targeted vaccination scheme introduced aimed at high risk individualseg. babies born in areas of UK where TB prevalence is high eg. London and ‘at risk’ employees eg. healthcare workers
What does Tuberculin Skin Testing (TST) / (Mantoux Test) demonstrate?
- Effective vaccination (BCG)
- Previous exposure to TB / infection with TB
Explain the principle of Tuberculin Skin Testing (TST) / (Mantoux Test)
TST detects delayed type 4 hypersensitivity reaction. If a previous TB infection has occurred sensitized T- lymphocytes circulate that respond to re-exposure to TB antigen to produce an injuration (indicative of a local inflammatory response)
Explain the procedure of Tuberculin Skin Testing (TST) / (Mantoux Test)
- Inject 0.1 ml of tuberculin purified protein derivative (PPD) into the inner surface of the forearm (intradermal injection).
- The injection should produce a pale elevation of the skin (wheal) 6 to 10 mm diameter
Explain the interpretation of Tuberculin Skin Testing (TST) / (Mantoux Test)
Examined between 48 and 72 hours; measure induration (mm); positive if the induration is >10mm.
NB. BCG vaccination generates a positive response
Generally, persons at high risk for developing TB disease fall into which two categories?
- Persons who have been recently infected with TB bacteria
- Persons with medical conditions that weaken the immune system
TB Risk Factors-Persons who have been Recently Infected with TB Bacteria
This includes:
- Close contacts of a person with infectious TB disease
- Persons who have immigrated from areas of the world with high rates of TB
- Children less than 5 years of age who have a positive TB test
- Groups with high rates of TB transmission, such as homeless persons, injection drug users, and persons with HIV infection
- Persons who work or reside with people who are at high risk for TB in facilities or institutions such as hospitals, homeless shelters, correctional facilities, nursing homes, and residential homes for those with HIV
TB Risk Factors
Babies and young children often have weak immune systems. Other people can have weak immune systems, too, especially people with any of these conditions:
- HIV infection (the virus that causes AIDS)
- Substance abuse
- Silicosis
- Diabetes mellitus
- Severe kidney disease
- Low body weight
- Organ transplants
- Head and neck cancer
- Medical treatments such as corticosteroids or organ transplant
- Specialized treatment for rheumatoid arthritis or Crohn’s disease
TB comorbidities: TB and HIV
- People living with HIV are from 15-22 times more likely to develop TB than persons without HIV.
- TB is the most common presenting illness among people living with HIV, including among those taking antiretroviral treatment and it is the major cause of HIV-related death.
- In 2018, there were an estimated 251 000 deaths from HIV-associated TB.
- Sub-Saharan Africa bears the brunt of the dual epidemic, accounting for approximately 84% of all TB/HIV deaths in 2018.
TB comorbidities: TB and diabetes
- The risk of TB among people with diabetes is 2-3 times higher than among those without diabetes.
- Diabetes can worsen the clinical course of TB, and TB can worsen glycaemic control in people with diabetes.
- Individuals with both conditions thus require careful clinical management.
- Strategies are needed to ensure that optimal care is provided to patients with both diseases.
- Diabetes prevalence is increasing globally.
- The most dramatic increase is in low- and middle income countries undergoing rapid economic, social, and lifestyle changes.
- Further increase in the number of diabetes-associated TB cases risks jeopardizing progress that has been made in the global fight against TB.
- Therefore, prevention and care of diabetes should be a priority not only for stakeholders involved in care and control of non-communicable diseases, but also for those working on TB care and prevention.
- This should be part of broader actions on risk factors and social determinants.
TB comorbidities: TB and nutrition
- Malnutrition increases the risk of TB and TB can lead to malnutrition.
- Malnutrition is therefore often highly prevalent among people with TB.
- While appropriate TB treatment often helps normalize nutritional status, many TB patients are still malnourished at the end of TB treatment.
- Therefore, nutritional assessment and counselling, and management of malnutrition based on the nutritional status are an important part of the TB treatment package.
- Undernutrition, and underlying food insecurity, are among the most important determinants of TB.
- Improving nutritional status at population level is important for TB prevention.
- This should be part of broader actions on social determinants.
TB comorbidities: TB and tobacco smoking
- Tobacco smoking increases the risk of TB 2-3 fold, and is associated with poor TB treatment results.
- Smoking prevalence is often high among people with TB, and prevalence of other smoking-related conditions can be high as well.
- People diagnosed with TB should be asked about smoking, and should be offered advice about smoking cessation.
- This is part of the practical approach to lung health.
- High smoking prevalence in the population is an important contributor to high TB burden. Public health and regulatory efforts to reduce smoking prevalence can have significant impact on TB incidence.
- It is key that TB prevention efforts are linked up with efforts to reduce other smoking related conditions in the population.
TB comorbidities: TB and harmful use of alcohol
- Harmful use of alcohol increases the risk of TB threefold, and is also a strong risk factor for poor TB treatment adherence.
- In countries with high prevalence of alcohol use disorders, and especially in intermediate- and low-incidence countries where TB has become highly concentrated to certain vulnerable groups, harmful alcohol use can be an important population level risk factor for TB, and is often a common co-morbidity among TB patients.
- As part of a comprehensive care package it is important, especially in those countries, to identify problem drinkers, diagnose alcohol use disorder, and refer for appropriate alcohol interventions.
- A few countries have experimented with systematic screening for harmful alcohol use of all TB patients.
- Screening and diagnosis of other mental health problems may also be warranted.
